B. burgdorferi polypeptides

ABSTRACT

Methods and compositions for the prevention, treatment and diagnosis of Lyme disease. Novel  B. burgdorferi  polypeptides, serotypic variants thereof, fragments thereof and derivatives thereof. Fusion proteins and multimeric proteins comprising same. Multicomponent vaccines comprising novel  B. burgdorferi  polypeptides in addition to other immunogenic  B. burgdorferi  polypeptides. DNA sequences, recombinant DNA molecules and transformed host cells useful in the compositions and methods. Antibodies directed against the novel  B. burgdorferi  polypeptides, and diagnostic kits comprising the polypeptides or antibodies.

This application claims priority under 35 U.S.C. § 120 as a divisionalof U.S. application Ser. No. 08/909,119, filed Aug. 11, 1997, now U.S.Pat. No. 5,807,685, which is a divisional of U.S. application Ser. No.08/118,469, filed Sep. 8, 1993, now U.S. Pat. No. 5,656,451, which is acontinuation in part of U.S. application Ser. No. 08/099,757, filed Jul.30, 1993, now abandoned.

TECHNICAL FIELD OF THE INVENTION

This invention relates to compositions and methods useful for theprevention, diagnosis and treatment of Lyme disease. More particularly,this invention relates to novel B. burgdorferi polypeptides which areable to elicit in a treated animal, the formation of an immune responsewhich is effective to prevent or lessen the severity, for some period oftime, of B. burgdorferi infection. This invention also relates tomulticomponent vaccines comprising one or more of the novel B.burgdorferi polypeptides. Also within the scope of this invention areantibodies directed against the novel B. burgdorferi polypeptides anddiagnostic kits comprising the antibodies or the polypeptides.

BACKGROUND OF THE INVENTION

Lyme borreliosis is the most common vector-borne infection in the UnitedStates [S. W. Barthold, et al., “An Animal Model For Lyme Arthritis”,Ann. N.Y. Acad. Sci., 539, pp. 264-73 (1988)]. It has been reported inevery continent except Antarctica. The clinical hallmark of Lyme Diseaseis an early expanding skin lesion known as erythema migrans, which maybe followed weeks to months later by neurologic, cardiac, and jointabnormalities.

The causative agent of Lyme disease is a spirochete known as Borreliaburgdorferi, transmitted primarily by Ixodes ticks of the Ixodes ricinuscomplex. B. burgdorferi has also been shown to be carried in otherspecies of ticks and in mosquitoes and deer flies, but it appears thatonly ticks of the I. ricinus complex are able to transmit the disease tohumans.

Lyme disease generally occurs in three stages. Stage one involveslocalized skin lesions (erythema migrans) from which the spirochete iscultured more readily than at any other time during infection [B. W.Berger et al., “Isolation And Characterization Of The Lyme DiseaseSpirochete From The Skin Of Patients With Erythema Chronicum Migrans”,J. Am. Acad. Dermatol., 3, pp. 444-49 (1985)]. Flu-like ormeningitis-like symptoms are common at this time. Stage two occurswithin days or weeks, and involves spread of the spirochete through thepatient's blood or lymph to many different sites in the body includingthe brain and joints. Varied symptoms of this disseminated infectionoccur in the skin, nervous system, and musculoskeletal system, althoughthey are typically intermittent. Stage three, or late infection, isdefined as persistent infection, and can be severely disabling. Chronicarthritis, and syndromes of the central and peripheral nervous systemappear during this stage, as a result of the ongoing infection andperhaps a resulting auto-immune disease [R. Martin et al., “Borreliaburgdorferi-Specific And Autoreactive T-Cell Lines From CerebrospinalFluid In Lyme Radiculomyelitis”, Ann Neurol., 24, pp. 509-16 (1988)].

B. burgdorferi is much easier to culture from the tick than from humans,therefore at present, Lyme disease is diagnosed primarily by serology.The enzyme-linked immunosorbent assay (ELISA) is one method ofdetection, using sonicated whole spirochetes as the antigen [J. E. Craftet al., “The Antibody Response In Lyme Disease: Evaluation Of DiagnosticTests”, J. Infect. Dis., 149, pp. 789-95 (1984)]. However, falsenegative and, more commonly, false positive results are associated withcurrently available tests.

At present, all stages of Lyme disease are treated with antibiotics.Treatment of early disease is usually effective, however the cardiac,arthritic, and nervous system disorders associated with the later stagesoften do not respond to therapy [A. C. Steere, “Lyme Disease”, New Eng.J. Med., 321, pp. 586-96 (1939)].

Like Treponema pallidum, which causes syphilis, and leptospirae, whichcause an infectious jaundice, Borrelia belong to the eubacterial phylumof spirochetes [A. G. Barbour and S. F. Hayes, “Biology Of BorreliaSpecies”, Microbiol. Rev., 50, pp. 381-400 (1986)]. Borrelia burgdorferihave a protoplasmic cylinder that is surrounded by a cell membrane, thenby flagella, and then by an outer membrane.

The B. burgdorferiouter surface proteins identified to date are believedto be lipoproteins, as demonstrated by labelling with [³H]palmitate [M.E. Brandt et al., “Immunogenic Integral membrane Proteins of Borreliaburgdorferi Are Lipoproteins”, Infect. Immun., 58, pp. 983-91 (1990)].The two major outer surface proteins are the 31 kd outer-surface proteinA (OspA) and the 34 kd outer surface protein B (OspB). Both proteinshave been shown to vary from different isolates or from differentpassages of the same isolate as determined by their molecular weightsand reactivity with monoclonal antibodies OspC is a 22 kDa membranelipoprotein previously identified as pC. [R. Fuchs et al., “MolecularAnalysis and Expression of a Borrelia burgdorferi Gene Encoding a 22 kDaProtein (pC) in Escherichia coli”, Mol. Microbiol., 6, pp. 503-09(1992)]. OspD is said to be preferentially expressed by low-passage,virulent strains of B. burgdorferi B31 [S. J. Norris et al.,“Low-Passage-Associated Proteins of Borrelia burgdorferi B31:Characterization and Molecular Cloning of OspD, A Surfaced-Exposed,Plasmid-Encoded Lipoprotein”, Infect. Immun., 60, pp. 4662-4672 (1992)].

Non-Osp B. burgdorferi proteins identified to date include the 41 kDflagellin protein, which is known to contain regions of homology withother bacterial flagellins [G. S. Gassman et al., “Analysis of theBorrelia burgdorferi GeHo fla Gene and Antigenic Characterization of ItsGene Product.”, J. Bacteriol., 173, pp. 1452-59 (1991)] and a 93kDaprotein said to be localized to the periplasmic space [D. J. Volkman etal., “Characterization of an Immunoreactive 93 kDa Core Protein ofBorrelia burgdorferi With a Human IgG Monoclonal Antibody”, J. Immun.,146, pp. 3177-82 (1991)].

Recently, immunization of mice with recombinant OspA has been shown tobe effective to confer long-lasting protection against subsequentinfection with B. burgdorferi [E. Fikrig et al., “Long-Term Protectionof Mice from Lyme Disease by Vaccination:with OspA”, Infec. Immun., 60,pp. 773-77 (1992)]. However, protection by the OspA immunogens used todate appears to be somewhat strain specific, probably due to theheterogeneity of the OspA gene among different B. burgdorferi isolates.For example, immunization with OspA from B. burgdorferi strain N40confers protection against subsequent infection with strains N40, B31and CD16, but not against strain 25015 [E. Fikrig et al., “Borreliaburgdorferi Strain 25015: Characterization of Outer Surface Protein Aand Vaccination Against Infection”, J. Immun., 148, pp. 2256-60 (1992)].

Immunization with OspB has also been shown to confer protection againstLyme disease but not to the same extent as that conferred by OspA [E.Fikrig et al., “Roles of OspA, OspB, and Flagellin in ProtectiveImmunity to Lyme Borreliosis in Laboratory Mice”, Infec. Immun., 60, pp.657-61 (1992)]. Moreover, some B. burgdorferi are apparently able toescape destruction in OspB-immunized mice via a mutation in the OspBgene which results in expression of a truncated OspB protein [E. Fikriget al., “Evasion of Protective Immunity by Borrelia burgdorferi byTruncation of Outer Surface Protein B”, Proc. Natl. Acad. Sci., 90, pp.4092-96 (1993)]. OspC has also been shown to have protective effects ina gerbil model of B. burgdorferi infection. However, the protectionafforded by immunization with this protein appears to be only partial[V. Preac-Mursic et al., “Active Immunization with pC Protein ofBorrelia burgdorferi Protects Gerbils against B. burgdorferi Infection”,Infection, 20, pp. 342-48 (1992)].

As prevention of tick infestation is imperfect, and Lyme disease may bemissed or misdiagnosed when it does appear, there exists a continuingurgent need for the determination of additional antigens of B.burgdorferi and related proteins which are able to elicit a protectiveimmune response and which may be useful in a broad-spectrum vaccine. Inaddition, identification of additional B. burgdorferi antigens mayenable the development of more reliable diagnostic reagents which areuseful in various stages of Lyme borreliosis.

DISCLOSURE OF THE INVENTION

The present invention provides novel B. burgdorferi polypeptides whichare substantially free of a B. burgdorferi spirochete or fragmentsthereof and which are thus useful in compositions and methods for thediagnosis, treatment and prevention of B. burgdorferi infection and Lymedisease. In one preferred embodiment, this invention provides OspEpolypeptides and pharmaceutically effective compositions and methodscomprising those polypeptides.

In another preferred embodiment, this invention provides OspFpolypeptides and pharmaceutically effective compositions and methodscomprising those polypeptides.

In another preferred embodiment, this invention provides S1 polypeptidesand pharmaceutically effective compositions and methods comprising thosepolypeptides.

In another preferred embodiment, this invention provides T5 polypeptidesand pharmaceutically effective compositions and methods comprising thosepolypeptides.

The preferred compositions and methods of each of the aforementionedembodiments are characterized by novel B. burgdorferi polypeptides whichelicit in treated animals, the formation of an immune response which iseffective to prevent or lessen the severity, for some period of time, ofB. burgdorferi infection.

In another preferred embodiment, this invention provides amulticomponent vaccine comprising one or more novel B. burgdorferipolypeptides of this invention in addition to one or more otherimmunogenic B. burgdorferi polypeptides. Such a vaccine is effective toconfer broad protection against B. burgdorferi infection.

In yet another embodiment, this invention provides antibodies directedagainst the novel B. burgdorferi polypeptides of this invention, andpharmaceutically effective compositions and methods comprising thoseantibodies.

In another embodiment, this invention provides diagnostic means andmethods characterized by one or more of the novel B. burgdorferipolypeptides, or antibodies directed against those polypeptides. Thesemeans and methods are useful for the detection of Lyme disease and B.burgdorferi infection. They are also useful in following the course oftreatment against such infection. In patients previously inoculated withthe vaccines of this invention, the detection means and methodsdisclosed herein are also useful for determining if booster inoculationsare appropriate.

In yet another embodiment, this invention provides methods foridentification and isolation of additional B. burgdorferi polypeptides,as well as compositions and methods comprising such polypeptides.

Finally, this invention provides DNA sequences that code for the novelB. burgdorferi polypeptides of this invention, recombinant DNA moleculesthat are characterized by those DNA sequences, unicellular hoststransformed with those DNA sequences and molecules, and methods of usingthose sequences, molecules and hosts to produce the novel B. burgdorferipolypeptides and multicomponent vaccines of this invention. DNAsequences of this invention are also advantageously used in methods andmeans for the diagnosis of Lyme disease and B. burgdorferi infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an immunoblot of protein extracts from cells transformedwith clone #11. The blot is probed with combined mouse anti-OspE andanti-OspF antiserum. Lane 1—uninduced clone #11; Lane 2—clone #11,induced; Lane 3—XL-1 Blue cells, uninduced.

FIG. 2 depicts a comparison of the control regions of transcription andtranslation among the DNA sequences encoding the novel B. burgdorferipolypeptides of this invention and the DNA sequences of other known B.burgdorferi outer surface proteins. The consensus “−35” and “−10” sigma70-like promoter sequences and consensus ribosomal binding sequence arefrom E. coli. The OspA and OspB sequences are from the OspA-B operon ofB. burgdorferi strain B31. OspC sequences are from strain PKo. OspDsequences are from strain B31.

FIG. 3 depicts the amino acid composition of the deduced OspE and OspFproteins. For each amino acid, the number outside the parenthesesindicates the total number of that particular amino acid; the numberinside the parentheses refers to the percent of the total amino acidsequence composed of that amino acid.

FIG. 4 depicts the codon usage of the OspE and OspF genes in B.burgdorferi N40. The preferred codons in B. burgdorferi OspA-B31,OspB-B31, OspC-PKo and OspD-B31 are underlined and bolded.

FIGS. 5 and 6 depict the hydrophilicity profiles of OspE and OspF,respectively, with a hydrophilicity window size of 7 and aKyte-Doolittle hydrophilicity scale.

FIG. 7 depicts a comparison of the N-terminal 30 amino acids of OspE andOspF. Identical amino acids are indicated with an asterisk. The cleavagesignal recognized by B. burgdorferi signal peptidase is underlined andshown in bold letters.

FIG. 8 depicts the separation of B. burgdorferi plasmid and chromosomalDNA by pulsed-field gel electrophoresis. The gel was visualized bystaining with ethidium bromide. Lane 1 contains the molecular weightstandard (concatemers of phage λ), lane 2 contains B. burgdorferi DNA.

FIG. 9 depicts Southern blots of several lanes of B. burgdorferi DNAseparated by pulsed-field gel electrophoresis and hybridized withvarious B. burgdorferi probes. Lane 1 is hybridized with a flagellinprobe, lane 2 with an OspD probe, lane 3 with an OspF probe and lane 4with an OspA probe.

FIG. 10 depicts an SDS-PAGE-gel of purified Δ20-OspE and Δ18-OspFpolypeptides. Lane 1 contains molecular weight markers, lane 2 containsΔ20-OspE polypeptide and lane 3 contains Δ18-OspF polypeptide.

FIG. 11 depicts an immunoblot of B. burgdorferi extracts probed withmouse anti-GT (control) sera (lane 1), mouse anti-OspE sera (lane 2) ormouse anti-OspF sera (lane 3). All of the sera were diluted 1:5000.

FIG. 12(A) depicts fixed B. burgdorferi spirochetes stained with rabbitantisera directed against Δ20-OspE. FIG. 12(B) depicts fixed B.burgdorferi spirochetes stained with rabbit antisera directed againstOspF.

FIG. 13 depicts three pairs of immunoblots of Δ20-OspE and Δ18-OspF. Theleft-hand pair of lanes depicts immunoblots of Δ20-OspE (lane 1) andΔ18-OspF (lane 2) probed with sera taken from mice 30 days afterinfection with B. burgdorferi. The center pair of lanes depictsimmunoblots of Δ20-OspE (lane 1) and Δ18-OspF (lane 2) probed with serataken from mice 90 days after infection with B. burgdorferi. Theright-hand pair of lanes depicts immunoblots of Δ20-OspE (lane 1) andΔ18-OspF (lane 2) probed with sera of a human patient with late-stageLyme disease.

FIG. 14 depicts the hydrophilicity profile of the T5 protein.

FIG. 15 depicts a coomasie-stained SDS-PAGE gel of the cleaved T5protein.

FIG. 16 Southern blots of several lanes of B. burgdorferi DNA separatedby pulsed-field gel electrophoresis and hybridized with various B.burgdorferi probes. Lane 1 is hybridized with a flagellin probe, lane 2with an OspD probe, lane 3 with a T5 probe.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to novel B. burgdorferi polypeptides and the DNAsequences which encode them, antibodies directed against thosepolypeptides, compositions comprising the polypeptides or antibodies,and methods for the detection, treatment and prevention of Lyme disease.

More specifically, in one embodiment, this invention relates tocompositions and methods comprising OspE polypeptides. The preferredcompositions and methods of this embodiment comprise immunogenic OspEpolypeptides that elicit in treated animals an immune response which iseffective to decrease the level of B. burgdorferi spirochetes in ticksfeeding on such animals.

In another embodiment, this invention relates to compositions andmethods comprising OspF polypeptides. The preferred compositions andmethods of this embodiment comprise immunogenic OspF polypeptides thatelicit in treated animals an immune response which is effective toprevent or lessen the severity, for some period of time, of B.burgdorferi infection. Immunogenic OspF polypeptides are not onlycapable of eliciting, in treated animals, an immune response which iseffective to decrease the level of B. burgdorferi spirochetes in ticksfeeding on such animals, but are also effective to protect the animalagainst B. burgdorferi infection and against Lyme disease-relateddisorders which would normally result from such infection.

In another embodiment, this invention relates to S1 polypeptides andpharmaceutically effective compositions and methods comprising thosepolypeptides.

In another embodiment, this invention relates to T5 polypeptides andpharmaceutically effective compositions and methods comprising thosepolypeptides.

The preferred compositions and methods of each of the aforementionedembodiments are characterized by novel B. burgdorferi polypeptides whichare also immunogenic B. burgdorferi polypeptides i.e., which elicit intreated animals, the formation of an immune response which is effectiveto prevent or lessen the severity, for some period of time, of B.burgdorferi infection.

In another embodiment, this invention relates to a multicomponentvaccine against Lyme disease comprising one or more of the novel B.burgdorferi polypeptides of this invention in addition to otherimmunogenic B. burgdorferi polypeptides. Such vaccine is useful toprotect against infection by a broad spectrum of B. burgdorferiorganisms.

All of the novel B. burgdorferi polypeptides provided by this invention,and the DNA sequences encoding them, are substantially free of a B.burgdorferi spirochete or fragments thereof, and thus may be used in avariety of applications without the risk of unintentional infection orcontamination with undesired B. burgdorferi components. Accordingly, thenovel. B. burgdorferi polypeptides of this invention are particularlyadvantageous in compositions and methods for the diagnosis andprevention of B. burgdorferi infection.

In another embodiment, this invention relates to compositions andmethods comprising antibodies directed against the novel B. burgdorferipolypeptides of this invention. Such antibodies may be used in a varietyof applications, including to detect the presence of B. burgdorferi, toscreen for expression of novel B. burgdorferi polypeptides, to purifynovel B. burgdorferi polypeptides, to block or bind to the novel B.burgdorferi polypeptides, to direct molecules to the surface of B.burgdorferi and to prevent or lessen the severity, for some period oftime, of B. burgdorferi infection.

In still another embodiment, this invention relates to diagnostic meansand methods characterized by the novel B. burgdorferi polypeptidesdisclosed herein or antibodies directed against those polypeptides.

In order to further define this invention, the following terms anddefinitions are herein provided.

As used herein, an “immunogenic B. burgdorferi polypeptide” is any B.burgdorferi molecule that, when administered to an animal, is capable ofeliciting an immune response that is effective to prevent or lessen theseverity, for some period of time, of B. burgdorferi infection.Preventing or lessening the severity of infection may be evidenced by achange in the physiological manifestations of erythema migrans,arthritis, carditis, neurological disorders, and other Lyme diseaserelated disorders. It may be evidenced by a decrease in or absenceof-spirochetes in the treated animal. And, it may be evidenced by adecrease in the level of spirochetes in infected ticks which have fed ontreated animals.

Immunogenic B. burgdorferi polypeptides are intended to include not onlythe novel B. burgdorferi polypeptides of this invention but also theOspA and OspB polypeptides disclosed in PCT patent application WO92/00055; the OspC protein as described in R. Fuchs et al., supra; otherB. burgdorferi proteins; and fragments, serotypic variants andderivatives of any of the above. In particular, immunogenic B.burgdorferi polypeptides are intended to include additional B.burgdorferi polypeptides which may also be identified according to themethods disclosed herein.

As used herein, a polypeptide which is “substantially free of a B.burgdorferi spirochete or fragments thereof” is a polypeptide that, whenintroduced into modified Barbour-Stoener-Kelly (BSK-II) medium andcultured at 37° C. for 7 days, fails to produce any B. burgdorferispirochetes detectable by dark field microscopy or a polypeptide that isdetectable as a single band on an immunoblot probed with polyclonalanti-B. burgdorferi anti-serum.

As used herein, an “OspE polypeptide” denotes a polypeptide which issubstantially free of B. burgdorferi spirochete or fragments thereof andwhich is selected from the group consisting of:

(a) an OspE protein consisting of amino acids 1-171 of SEQ ID NO: 2 andserotypic variants thereof;

(b) fragments comprising at least 8 amino acids taken as a block fromthe OspE polypeptide of (a);

(c) derivatives of an OspE polypeptide of (a) or (b), said derivativesbeing at least 80% identical in amino acid sequence to the correspondingpolypeptide of (a) or (b);

(d) polypeptides that are immunologically reactive with antibodiesgenerated by infection of a mammalian host with B. burgdorferi, whichantibodies are immunologically reactive with an OspE polypeptide of (a)or (b) or (c); (e) polypeptides that are capable of eliciting antibodiesthat are immunologically reactive with B. burgdorferi and the OspEpolypeptide of (a) or (b) or (c); and

(f) polypeptides that are immunologically reactive with antibodieselicited by immunization with the OspE polypeptide of (a) or (b) or (c).

As used herein, a “serotypic variant” of an OspE polypeptide is anynaturally occurring polypeptide which may be encoded in whole or inpart, by a DNA sequence which hybridizes, at 20-27° C. below Tm, to theDNA sequence encoding the OspE protein of SEQ ID NO: 2. One of skill inthe art will understand that serotypic variants of an OspE polypeptideinclude polypeptides encoded by DNA sequences of which any portion maybe amplified by using the polymerase chain reaction and oligonucleotideprimers derived from any portion of the DNA sequence encoding the OspEprotein of SEQ ID NO: 2.

As used herein, an “OspF polypeptide” denotes a polypeptide which issubstantially free of a B. burgdorferi spirochete or fragments thereofand which is selected from the group consisting of:

(a) an OspF protein consisting of amino acids 1-230 of SEQ ID NO: 3 andserotypic variants thereof;

(b) fragments comprising at least 8 amino acids taken as a block fromthe OspF polypeptide of (a);

(c) derivatives of the OspF polypeptide of (a) or (b), said derivativesbeing at least 80% identical in amino acid sequence to the correspondingpolypeptide of (a) or (b);

(d) polypeptides that are immunologically reactive with antibodiesgenerated by infection of a mammalian host with B. burgdorferi, whichantibodies are immunologically reactive with an OspF polypeptide of (a)or (b) or (c);

(e) polypeptides that are capable of eliciting antibodies that areimmunologically reactive with B. burgdorferi and the OspF polypeptide of(a) or (b) or (c); and

(f) polypeptides that are immunologically reactive with antibodieselicited by immunization with the OspF polypeptide of (a) or (b) or (c).

As used herein, a “serotypic variant” of an OspF polypeptide is anynaturally occurring polypeptide which may be encoded, in whole or inpart, by a DNA sequence which hybridizes, at 20-27° C. below Tm, to theDNA sequence encoding the OspF protein of SEQ ID NO: 3. As withserotypic variants of OspE polypeptides, one of skill in the art willreadily appreciate that serotypic variants of OspF polypeptides includethose polypeptides encoded by B. burgdorferi DNA sequences of which anyportion may be amplified by using the polymerase chain reaction andoligonucleotide primers derived from any portion of the DNA sequenceencoding the OspF protein of SEQ ID NO: 3.

As used herein, an “S1 polypeptide” denotes a polypeptide which issubstantially free of a B. burgdorferi spirochete or fragments thereofand which is selected from the group consisting of:

(a) an S1 protein having the amino acid sequence of SEQ ID NO: 5 andserotypic variants thereof;

(b) fragments comprising at least 8 amino acids taken as a block fromthe S1 polypeptide of (a);

(c) derivatives of the S1 polypeptide of (a) or (b), said derivativesbeing at least 80% identical in amino acid sequence to the correspondingpolypeptide of (a) or (b);

(d) polypeptides that are immunologically reactive with antibodiesgenerated by infection of a mammalian host with B. burgdorferi, whichantibodies are immunologically reactive with an S1 polypeptide of (a) or(b) or (c);

(e) polypeptides that are capable of eliciting antibodies that areimmunologically reactive with B. burgdorferi and the S1 polypeptide of(a) or (b) or (c); and

(f) polypeptides that are immunologically reactive with antibodieselicited by immunization with the S1 polypeptide of (a) or (b) or (c).

As used herein, a “serotypic variant” of an S1 polypeptide is anynaturally occurring polypeptide which may be encoded, in whole or inpart, by a DNA sequence which hybridizes, at 20-27° C. below Tm, to theDNA sequence encoding the S1 protein SEQ ID NO: 5. Again, serotypicvariants of S1 polypeptides include those polypeptides encoded by B.burgdorferi DNA sequences of which any portion may be amplified by usingthe polymerase chain reaction and oligonucleotide primers derived fromany portion of the DNA sequence encoding the S1 protein of SEQ ID NO: 5.

As used herein, a “T5 polypeptide” denotes a polypeptide which issubstantially free of a B. burgdorferi spirochete or fragments thereofand which is selected from the group consisting of:

(a) a T5 polypeptide having the amino acid sequence of SEQ ID NO: 7 andserotypic variants thereof;

(b) fragments comprising at least 8 amino acids taken as a block fromthe T5 polypeptide of (a);

(c) derivatives of the T5 polypeptide of (a) or (b) said derivativesbeing at least 80% identical in amino acid sequence to the correspondingpolypeptide of (a) or (b);

(d) polypeptides that are immunologically-reactive with antibodiesgenerated by infection of a mammalian host with B. burgdorferi, whichantibodies are immunologically reactive with a T5 polypeptide of (a) or(b) or (c);

(e) polypeptides that are capable of eliciting antibodies that areimmunologically reactive with B. burgdorferi and the T5 polypeptide of(a) or (b) or (c); and

(f) polypeptides that are immunologically reactive with antibodieselicited by immunization with the T5 polypeptide of (a) or (b) or (c).

As used herein, a “serotypic variant” of a T5 polypeptide is anynaturally occurring polypeptide which may be encoded, in whole or inpart, by a DNA sequence which hybridizes, at 20-27° C. below Tm, to theDNA sequence encoding the T5 protein SEQ ID NO: 7. Again, serotypicvariants of T5 polypeptides include those polypeptides encoded by B.burgdorferi DNA sequences of which any portion may be amplified by usingthe polymerase chain reaction and oligonucleotide primers derived fromany portion of the DNA sequence encoding the T5 protein of SEQ ID NO: 7.

As used herein, a “novel B. burgdorferi polypeptide” is an OspEpolypeptide, an OspF polypeptide, an S1 polypeptide, a T5 polypeptide,or one or more B. burgdorferi polypeptides encoded in whole or in partby a DNA sequence present in clone 4, 5 or 7 as described in Example XV,infra.

As used herein, a “derivative” a novel B. burgdorferi polypeptide is apolypeptide in which one or more physical, chemical, or biologicalproperties has been altered. Such modifications include, but are notlimited to: amino acid substitutions, modifications, additions ordeletions; alterations in the pattern of lipidation, glycosylation orphosphorylation; reactions of free amino, carboxyl, or hydroxyl sidegroups of the amino acid residues present in the polypeptide with otherorganic and non-organic molecules; and other modifications, any of whichmay result in changes in primary, secondary or tertiary structure.

As used herein, a “protective antibody” is an antibody that confersprotection, for some period of time, against any one of thephysiological disorders associated with B. burgdorferi infection.

As used herein, a “protective epitope” is (1) an epitope which isrecognized by a protective antibody, and/or (2) an epitope which, whenused to immunize an animal, elicits an immune response sufficient toprevent or lessen the severity for some period of time, of B.burgdorferi infection. Again, preventing or lessening the severity ofinfection may be evidenced by a change in the physiologicalmanifestations of erythema migrans, arthritis, carditis, neurologicaldisorders, and other Lyme disease related disorders. It may be evidencedby a decrease in the level of spirochetes in the treated animal. And, itmay also be evidenced by a decrease in the level of spirochetes ininfected ticks feeding on treated animals. A protective epitope maycomprise a T cell epitope, a B cell epitope, or combinations thereof.

As used herein, a “T cell epitope” is an epitope which, when presentedto T cells by antigen presenting cells, results in a T cell responsesuch as clonal expansion or expression of lymphokines or otherimmunostimulatory molecules. A T cell epitope may also be an epitoperecognized by cytotoxic T cells that may affect intracellular B.burgdorferi infection. A strong T cell epitope is a T cell epitope whichelicits a strong T cell response.

As used herein, a “B cell epitope” is the simplest spatial conformationof an antigen which reacts with a specific antibody.

As used herein, a “therapeutically effective amount” of a polypeptide orof an antibody is the amount that, when administered to an animal,elicits an immune response that is effective to prevent or lessen theseverity, for some period of time, of B. burgdorferi infection.

As used herein, an “anti-OspE polypeptide antibody” is an immunoglobulinmolecule or portion thereof, that is immunologically reactive with anOspE polypeptide of the present invention, and that was either elicitedby immunization with an OspE polypeptide of this invention or wasisolated or identified by its reactivity with an OspE polypeptide ofthis invention.

As used herein, an “anti-OspF polypeptide antibody” is an immunoglobulinmolecule, or portion thereof, that is immunologically reactive with anOspF polypeptide of the present invention and that was either elicitedby immunization with an OspF polypeptide of this invention or wasisolated or identified by its reactivity with an OspF polypeptide ofthis invention.

As used herein an “anti-S1 polypeptide antibody” is an immunoglobulinmolecule, or portion thereof, that is immunologically reactive with anS1 polypeptide of the present invention and that was either elicited byimmunization with an S1 polypeptide of this invention or was isolated oridentified by its reactivity with an S1 polypeptide of this invention.

As used herein an “anti-T5 polypeptide antibody” is an immunoglobulinmolecule, or portion thereof, that is immunologically reactive with a T5polypeptide of the present invention and that was either elicited byimmunization with a T5 polypeptide of this invention or was isolated oridentified by its reactivity with a T5 polypeptide of this invention.

As used herein, an “antibody directed against a novel B. burgdorferipolypeptide” (also referred to as “an antibody of this invention”) is ananti-OspE polypeptide antibody, an anti-OspF polypeptide antibody, ananti-S1 polypeptide antibody or an anti-T5 polypeptide antibody. Itshould be understood that an antibody directed against a novel B.burgdorferi polypeptide may also be a protective antibody.

It should also be understood that the antibodies of this invention arenot intended to include those antibodies which are normally elicited inan animal upon infection with naturally occurring B. burgdorferi andwhich have not been removed from or altered within the animal in whichthey were elicited.

An antibody directed against a novel B. burgdorferi polypeptide may bean intact immunoglobulin molecule or a portion of an immunoglobulinmolecule that contains an intact antigen binding site, including thoseportions known in the art as F(v), Fab, Fab′ and F(ab′)2. It may also bea genetically engineered or synthetically produced molecule.

The novel B. burgdorferi polypeptides disclosed herein areimmunologically reactive with antisera generated by infection of amammalian host with B. burgdorferi. Accordingly, they are useful inmethods and compositions to diagnose and protect against Lyme disease,and in therapeutic compositions to stimulate immunological clearance ofB. burgdorferi during ongoing infection. In addition, because at leastsome, if not all of the novel B. burgdorferi polypeptides disclosedherein are immunogenic surface proteins of B. burgdorferi, they areparticularly useful in a multicomponent vaccine against Lyme disease,because such a vaccine may be formulated to more closely resemble theimmunogens presented by replication-competent B. burgdorferi, andbecause such a vaccine is more likely to confer broad-spectrumprotection than a vaccine comprising only a single B. burgdorferipolypeptide. Multicomponent vaccines according to this invention mayalso contain polypeptides which characterize any currently existing orto be discovered vaccine useful for immunization of diseases other thanLyme disease such as, for example, diphtheria, polio, hepatitis, andmeasles. Such multicomponent vaccines are characterized by a singlecomposition form.

The preferred compositions and methods of this invention comprise novelB. burgdorferi polypeptides having enhanced immunogenicity. Suchpolypeptides may result when the native forms of the polypeptides orfragments thereof are modified or subjected to treatments to enhancetheir immunogenic character in the intended recipient.

Numerous techniques are available and well known to those of skill inthe art which may be used, without undue experimentation, tosubstantially increase the immunogenicity of the novel B. burgdorferipolypeptides herein disclosed. For example, the polypeptides may bemodified by coupling to dinitrophenol groups or arsanilic acid, or bydenaturation with heat and/or SDS.

Particularly if the polypeptides are small polypeptides synthesizedchemically, it may be desirable to couple them to an immunogeniccarrier. The coupling of course, must not interfere with the ability ofeither the polypeptide or the carrier to function appropriately. For areview of some general considerations in coupling strategies, seeAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E.Harlow and D. Lane (1988). Useful immunogenic carriers are well known inthe art. Examples of such carriers are keyhole limpet hemocyanin (KLH);albumins such as bovine serum albumin (BSA) and ovalbumin, PPD (purifiedprotein derivative of tuberculin); red blood cells; tetanus toxoid;cholera toxoid; agarose beads.; activated carbon; or bentonite.

Modification of the amino acid sequence of the novel B. burgdorferipolypeptides disclosed herein in order to alter the lipidation state isalso a method which may be used to increase their immunogenicity andbiochemical properties. For example, the polypeptides or fragmentsthereof may be expressed with or without the signal sequences thatdirect addition of lipid moieties.

As will be apparent from the disclosure to follow, the polypeptides mayalso be prepared with the objective of increasing stability or renderingthe molecules more amenable to purification and preparation. One suchtechnique is to express the polypeptides as fusion proteins comprisingother B. burgdorferi or non-B. burgdorferi sequences.

In accordance with this invention, derivatives of the novel B.burgdorferi polypeptides may be prepared by a variety of methods,including by in vitro manipulation of the DNA encoding the nativepolypeptides and subsequent expression of the modified DNA, by chemicalsynthesis of derivatized DNA sequences, or by chemical or biologicalmanipulation of-expressed amino acid sequences.

For example, derivatives may be produced by substitution of one or moreamino acids with a different natural amino acid, an amino acidderivative or non-native amino acid, conservative substitution beingpreferred, e.g., 3-methylhistidine may be substituted for histidine,4-hydroxyproline may be substituted for proline, 5-hydroxylysine may besubstituted for lysine, and the like.

Causing amino acid substitutions which are less conservative may alsoresult in desired derivatives, e.g., by causing changes in charge,conformation and other biological properties. Such substitutions wouldinclude for example, substitution of a hydrophilic residue for ahydrophobic residue, substitution of a cysteine or proline for anotherresidue, substitution of a residue having a small side chain for aresidue having a bulky side chain or substitution of a residue having anet positive charge for a residue having a net negative charge. When theresult of a given substitution cannot be predicted with certainty, thederivatives may be readily assayed according to the methods disclosedherein to determine the presence or absence of the desiredcharacteristics.

In a preferred embodiment of this invention, the novel B. burgdorferipolypeptides disclosed herein are prepared as part of a larger fusionprotein. For example, a novel B. burgdorferi polypeptide of thisinvention may be fused at its N-terminus or C-terminus to a differentimmunogenic B. burgdorferi polypeptide, to a non-B. burgdorferipolypeptide or to combinations thereof, to produce fusion proteinscomprising the novel B. burgdorferi polypeptide.

In a preferred embodiment of this invention, fusion proteins comprisingnovel B. burgdorferi polypeptides are constructed comprising B celland/or T cell epitopes from multiple serotypic variants of B.burgdorferi, each variant differing from another with respect to thelocations or sequences of the epitopes within the polypeptide. In a morepreferred embodiment, fusion proteins are constructed which comprise oneor more of the novel B. burgdorferi polypeptides fused to otherimmunogenic B. burgdorferi polypeptides. Such fusion proteins areparticularly effective in the prevention, treatment and diagnosis ofLyme disease as caused by a wide spectrum of B. burgdorferi isolates.

In another preferred embodiment of this invention, the novel B.burgdorferi polypeptides are fused to moieties, such as immunoglobulindomains, which may increase the stability and prolong the in vivo plasmahalf-life of the polypeptide. Such fusions may be prepared according tomethods well known to those of skill in the art, for example, inaccordance with the teachings of U.S. Pat. No. 4,946,778, or U.S. Pat.No. 5,116,964. The exact site of the fusion is not critical as long asthe polypeptide retains the desired biological activity. Suchdeterminations may be made according to the teachings herein or by othermethods known to those of skill in the art.

It is preferred that the fusion proteins comprising the novel B.burgdorferi polypeptides be produced at the DNA level, e.g., byconstructing a nucleic acid molecule encoding the fusion, transforminghost cells with the molecule, inducing the cells to express the fusionprotein, and recovering the fusion protein from the cell culture.Alternatively, the fusion proteins may be produced after gene expressionaccording to known methods.

The novel B. burgdorferi polypeptides may also be part of largermultimeric molecules which may be produced recombinantly or may besynthesized chemically. Such multimers may also include the polypeptidesfused or coupled to moieties other than amino acids, including lipidsand carbohydrates.

Preferably, the multimeric proteins will consist of multiple T or B cellepitopes or combinations thereof repeated within the same molecule,either randomly, or with spacers (amino acid or otherwise) between them.

In the most preferred embodiment of this invention, the novel B.burgdorferi polypeptides of this invention which are also immunogenic B.burgdorferi polypeptides are incorporated into a multicomponent vaccinewhich also comprises other immunogenic B. burgdorferi polypeptides. Sucha multicomponent vaccine, by virtue of its ability to elicit antibodiesto a variety of immunogenic B. burgdorferi polypeptides, will beeffective to protect against Lyme disease as caused by a broad spectrumof different B. burgdorferi isolates, even those that may not expressone or more of the Osp proteins.

The multicomponent vaccine may contain the novel B. burgdorferipolypeptides as part of a multimeric molecule in which the variouscomponents are covalently associated. Alternatively, it may containmultiple individual components. For example, a multicomponent vaccinemay be prepared comprising two or more of the novel B. burgdorferipolypeptides, or comprising one novel B. burgdorferi polypeptide and onepreviously identified B. burgdorferi polypeptide, wherein eachpolypeptide is expressed and purified from independent cell cultures andthe polypeptides are combined prior to or during formulation.

Alternatively, a multicomponent vaccine may be prepared fromheterodimers or tetramers wherein the polypeptides have been fused toimmunoglobulin chains or portions thereof. Such a vaccine couldcomprise, for example, an OspF polypeptide fused to an immunoglobulinheavy chain and an OspA polypeptide fused to an immunoglobulin lightchain, and could be produced by transforming a host cell with DNAencoding the heavy chain fusion and DNA encoding the light chain fusion.One of skill in the art will understand that the host cell selectedshould be capable of assembling the two chains appropriately.Alternatively, the heavy and light chain fusions could be produced fromseparate cell lines and allowed to associate after purification.

The desirability of including a particular component-and the relativeproportions of each component may be determined by using the assaysystems disclosed herein, or by using other systems known to those inthe art. Most preferably, the multicomponent vaccine will comprisenumerous T cell and B cell epitopes of immunogenic B. burgdorferipolypeptides, including the novel B. burgdorferi polypeptides of thisinvention.

This invention also contemplates that the novel B. burgdorferipolypeptides of this invention, either alone or with other immunogenicB. burgdorferi polypeptides, may be administered to an animal via aliposome delivery system in order to enhance their stability and/orimmunogenicity. Delivery of the novel B. burgdorferi polypeptides vialiposomes may be particularly advantageous because the liposome may beinternalized by phagocytic cells in the treated animal. Such cells, uponingesting the liposome, would digest the liposomal membrane andsubsequently present the polypeptides to the immune system inconjunction with other molecules required to elicit a strong immuneresponse.

The liposome system may be any variety of unilamellar vesicles,multilamellar vesicles, or stable plurilamellar vesicles, and may beprepared and administered according to methods well known to those ofskill in the art, for example in accordance with the teachings of U.S.Pat. Nos. 5,169,637, 4,762,915, 5,000,958 or 5,185,154. In addition, itmay be desirable to express the novel B. burgdorferi polypeptides ofthis invention, as well as other selected B. burgdorferi polypeptides,as lipoproteins, in order to enhance their binding to liposomes.

Any of the novel B. burgdorferi polypeptides of this invention may beused in the form of a pharmaceutically acceptable salt. Suitable acidsand bases which are capable of forming salts with the polypeptides ofthe present invention are well known to those of skill in the art, andinclude inorganic and organic acids and bases.

According to this invention, we describe a method which comprises thesteps of treating an animal with a therapeutically effective amount of anovel B. burgdorferi polypeptide, or a fusion protein or a multimericprotein comprising a novel B. burgdorferi polypeptide, in a mannersufficient to prevent or lessen the severity, for some period of time,of B. burgdorferi infection. The polypeptides that are preferred for usein such methods are those that contain protective epitopes. Suchprotective epitopes may be B cell epitopes, T cell epitopes, orcombinations thereof.

According to another embodiment of this invention, we describe a methodwhich comprises the steps of treating an animal with a multicomponentvaccine comprising a therapeutically effective amount of a novel B.burgdorferi polypeptide, or a fusion protein or multimeric proteincomprising such polypeptide in a manner sufficient to prevent or lessenthe severity, for some period of time, of B. burgdorferi infection.Again, the polypeptides, fusion proteins and multimeric proteins thatare preferred for use in such methods are those that contain protectiveepitopes, which may be B cell epitopes, T cell epitopes, or combinationsthereof.

The most preferred polypeptides, fusion proteins and multimeric proteinsfor use in these compositions and methods are those containing bothstrong T cell and B cell epitopes. Without being bound by theory, webelieve that this is the best way to stimulate high titer antibodiesthat are effective to neutralize B. burgdorferi infection. Suchpreferred polypeptides will be internalized by B cells expressingsurface immunoglobulin that recognizes the B cell epitope(s). The Bcells will then process the antigen and present it to T cells. The Tcells will recognize the T cell epitope(s) and respond by proliferatingand producing lymphokines which in turn cause B cells to differentiateinto antibody producing plasma cells. Thus, in this system, a closedautocatalytic circuit exists which will result in the amplification ofboth B and T cell responses, leading ultimately to production of astrong immune response which includes high titer antibodies against thenovel B. burgdorferi polypeptide.

One of skill in the art will also understand that it may be advantageousto administer the novel B. burgdorferi polypeptides of this invention ina form that will favor the production of T-helper cells type 2 (T_(H)2),which help B cells to generate antibody responses. Aside fromadministering epitopes which are strong B cell epitopes, the inductionof T_(H)2 cells may also be favored by the mode of administration of thepolypeptide for example by administering in certain doses or withparticular adjuvants and immunomodulators, for example withinterleukin-4.

To prepare the preferred polypeptides of this invention, in oneembodiment, overlapping fragments of the novel B. burgdorferipolypeptides of this invention are constructed. The polypeptides thatcontain B cell epitopes may be identified in a variety of ways forexample by their ability to (1) remove protective antibodies frompolyclonal antiserum directed against the polypeptide or (2) elicit animmune response which is effective to prevent or lessen the severity ofB. burgdorferi infection.

Alternatively, the polypeptides may be used to produce monoclonalantibodies which could be screened for their ability to conferprotection against B. burgdorferi infection when used to immunize naiveanimals. Once a given monoclonal antibody is found to confer protection,the particular epitope that is recognized by that antibody may then beidentified.

As recognition of T cell epitopes is MHC restricted, the polypeptidesthat contain T cell epitopes may be identified in vitro by testing themfor their ability to stimulate proliferation and/or cytokine productionby T cell clones generated from humans of various HLA types, from thelymph nodes of C3H/He mice, or from domestic animals. Compositionscomprising multiple T cell epitopes recognized by individuals withdifferent Class II antigens are useful for prevention and treatment ofLyme disease in a broad spectrum of patients.

In a preferred embodiment of the present invention, a novel B.burgdorferi polypeptide containing a B cell epitope is fused to one ormore other immunogenic B. burgdorferi polypeptides containing strong Tcell epitopes. The fusion protein that carries both strong T cell and Bcell epitopes is able to participate in elicitation of a high titerantibody response effective to neutralize infection with B. burgdorferi.

Strong T cell epitopes may also be provided by non-B. burgdorferimolecules. For example, strong T cell epitopes have been observed inhepatitis B virus core antigen (HBcAg). Furthermore, it has been shownthat linkage of one of these segments to segments of the surface antigenof Hepatitis B virus, which are poorly recognized by T cells, results ina major amplification of the anti-HBV surface antigen response, [D. R.Milich et al., “Antibody Production To The Nucleocapsid And Envelope OfThe Hepatitis B Virus Primed By A Single Synthetic T Cell Site”, Nature,329, pp. 547-49 (1987)].

Therefore, in yet another preferred embodiment, B cell epitopes of thenovel B. burgdorferi polypeptides are fused to segments of HBcAG or toother antigens which contain strong T cell epitopes, to produce a fusionprotein that can elicit a high titer antibody response against B.burgdorferi. In addition, it may be particularly advantageous to link anovel B. burgdorferi polypeptide of this invention to a strong immunogenthat is also widely recognized, for example tetanus toxoid.

It will be readily appreciated by one of ordinary skill in the art thatthe novel B. burgdorferi polypeptides of this invention, as well asfusion proteins and multimeric proteins containing them, may be preparedby recombinant means, chemical means, or combinations thereof.

For example, the polypeptides may be generated by recombinant meansusing the DNA sequences of B. burgdorferi strain N40 as set forth in thesequence listings contained herein. DNA encoding serotypic variants ofthe polypeptides may likewise be cloned, e.g., using PCR andoligonucleotide primers derived from the sequences herein disclosed.

In this regard, it may be particularly desirable to isolate the genesencoding novel B. burgdorferi polypeptides from strain 25015 and otherstrains of B. burgdorferi that are known to differ antigenically fromstrain N40, in order to obtain a broad spectrum of different epitopeswhich would be useful in the methods and compositions of this invention.For example, the OspA gene of B. burgdorferi strain 25015 is known todiffer from the OspA gene of B. burgdorferi strain N40 to the extentthat anti-OspA antibodies, which protect against subsequent infectionwith strain N40, appear ineffective to protect against infection withstrain 25015.

Oligonucleotide primers and other nucleic acid probes derived from thegenes encoding the novel B. burgdorferi polypeptides may also be used toisolate and clone other related surface proteins from B. burgdorferi andrelated spirochetes which may contain regions of DNA sequence homologousto the DNA sequences of this invention. In addition, the DNA sequencesof this invention may also be used in PCR reactions to detect thepresence of B. burgdorferi in a suspected infected sample.

If the novel B. burgdorferi polypeptides of this invention are producedrecombinantly, they may be expressed in unicellular hosts. As is wellknown to one of skill in the art, in order to obtain high expressionlevels of foreign DNA sequences in a host, the sequences are generallyoperatively linked to transcriptional and translational expressioncontrol sequences that are functional in the chosen host. Preferably,the expression control sequences, and the gene of interest, will becontained in an expression vector that further comprises a selectionmarker.

The DNA sequences encoding the polypeptides of this invention may or maynot encode a signal sequence. If the expression host is eukaryotic, itgenerally is preferred that a signal sequence be encoded so that themature protein is secreted from the eukaryotic host.

An amino terminal methionine may or may not be present on the expressedpolypeptides of this invention. If the terminal methionine is notcleaved by the expression host, it may, if desired, be chemicallyremoved by standard techniques.

A wide variety of expression host/vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors for eukaryotic hosts, include, for example, vectors comprisingexpression control sequences from SV40, bovine papilloma virus,adenovirus, adeno-associated virus, cytomegalovirus and retroviruses.Useful expression vectors for bacterial hosts include bacterialplasmids, such as those from E. coli, including pBluescript, pGEX-2T,pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives, widerhost range plasmids, such as RP4, phage DNAS, e.g., the numerousderivatives of phage lambda, e.g. λGT10 and λGT11, and other phages.Useful expression vectors for yeast cells include the 2μ plasmid andderivatives thereof. Useful vectors for insect cells include pVL 941.

In addition, any of a wide variety of expression controlsequences—sequences that control the expression of a DNA sequence whenoperatively linked to it—may be used in these vectors to express the DNAsequences of this invention. Such useful expression control sequencesinclude the expression control sequences associated with structuralgenes of the foregoing expression vectors. Examples of useful expressioncontrol sequences include, for example, the early and late promoters ofSV40 or adenovirus, the lac system, the trp system, the TAC or TRCsystem, the T3 and T7 promoters, the major operator and promoter regionsof phage lambda, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, the promoters of the yeast α-matingsystem and other constitutive and inducible promoter sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

In a preferred embodiment, DNA sequences encoding the novel B.burgdorferi polypeptides of this invention are cloned in the expressionvector lambda ZAP II (Stratagene, La Jolla, Calif.), in which expressionfrom the lac promoter may be induced by IPTG.

In another preferred embodiment, DNA encoding the novel B. burgdorferipolypeptides of this invention is inserted in frame into an expressionvector that allows high level expression of the polypeptide as aglutathione S-transferase fusion protein. Such a fusion protein thuscontains amino acids encoded by the vector sequences as well as aminoacids of the novel. B. burgdorferi polypeptide.

A wide variety of unicellular host cells are useful in expressing theDNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, streptomyces, fungi, yeast, insect cells such asSpodoptera frugiperda (SF9), animal cells such as CHO and mouse cells,African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT10, and human cells, as well as plant cells in tissue culture.

It should of course be understood that not all vectors and expressioncontrol sequences will function equally well to express the DNAsequences of this invention. Neither will all hosts function equallywell with the same expression system. However, one of skill in the artmay make a selection among these vectors, expression control sequencesand hosts without undue experimentation and without departing from thescope of this invention. For example, in selecting a vector, the hostmust be considered because the vector must be replicated in it. Thevector's copy number, the ability to control that copy number, theability to control integration, if any, and the expression of any otherproteins encoded by the vector, such as antibiotic or other selectionmarkers, should also be considered.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe sequence, its controllability, and its compatibility with the DNAsequence of this invention, particularly with regard to potentialsecondary structures. Unicellular hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded for by the DNA sequences of thisinvention, their secretion characteristics, their ability to fold thepolypeptide correctly, their fermentation or culture requirements, andthe ease of purification from them of the products coded for by the DNAsequences of this invention.

Within these parameters, one of skill in the art may select variousvector/expression control sequence/host combinations that will expressthe DNA sequences of this invention on fermentation or in other largescale cultures.

The molecules comprising the novel B. burgdorferi polypeptides encodedby the DNA sequences of this invention may be isolated from thefermentation or cell culture and purified using any of a variety ofconventional methods including: liquid chromatography such as normal orreversed phase, using HPLC, FPLC and the like; affinity chromatography(such as with inorganic ligands or monoclonal antibodies); sizeexclusion chromatography; immobilized metal chelate chromatography; gelelectrophoresis; and the like. One of skill in the art may select themost appropriate isolation and purification techniques without departingfrom the scope of this invention.

In addition, the novel B. burgdorferi polypeptides may be generated byany of several chemical techniques. For example, they may be preparedusing the solid-phase synthetic technique originally described by R. B.Merrifield, “Solid Phase Peptide Synthesis. I. The Synthesis Of ATetrapeptide”, J. Am. Chem. Soc., 83, p. 2149-54 (1963), or they may beprepared by synthesis in solution. A summary of peptide synthesistechniques may be found in E. Gross & H. J. Meinhofer, 4 The Peptides:Analysis, Synthesis, Biology; Modern Techniques Of Peptide And AminoAcid Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles ofPeptide Synthesis, Springer-Verlag (1984).

Typically, these synthetic methods comprise the sequential addition ofone or more amino acid residues to a growing peptide chain. Oftenpeptide coupling agents are used to facilitate this reaction. For arecitation of peptide coupling agents suitable for the uses describedherein see M. Bodansky, supra. Normally, either the amino or carboxylgroup of the first amino acid residue is protected by a suitable,selectively removable protecting group. A different protecting group isutilized for amino acids containing a reactive side group, e.g., lysine.A variety of protecting groups known in the field of peptide synthesisand recognized by conventional abbreviations therein, may be found in T.Greene, Protective Groups In Organic Synthesis, Academic Press (1981).

According to another embodiment of this invention, antibodies directedagainst the novel B. burgdorferi polypeptides are generated. Suchantibodies are immunoglobulin molecules or portions thereof that areimmunologically reactive with a novel B. burgdorferi polypeptide of thepresent invention. It should be understood that the antibodies of thisinvention include antibodies immunologically reactive with fusionproteins and multimeric proteins comprising a novel B. burgdorferipolypeptide.

Antibodies directed against a novel B. burgdorferi polypeptide may begenerated by infection of a mammalian host with B. burgdorferi, or byimmunization of a mammalian host with a novel B. burgdorferi polypeptideof the present invention. Such antibodies may be polyclonal ormonoclonal, it is preferred that they are monoclonal. Methods to producepolyclonal and monoclonal antibodies are well known to those of skill inthe art. For a review of such methods, see Antibodies, A LaboratoryManual, supra, and D. E. Yelton, et al., Ann. Rev. of Biochem., 50, pp.657-80 (1981). Determination of immunoreactivity with a novel B.burgdorferi polypeptide of this invention may be made by any of severalmethods well known in the art, including by immunoblot assay and ELISA.

An antibody of this invention may also be a hybrid molecule formed fromimmunoglobulin sequences from different species (e.g., mouse and human)or from portions of immunoglobulin light and heavy chain sequences fromthe same species. It may be a molecule that has multiple bindingspecificities, such as a bifunctional antibody prepared by any one of anumber of techniques known to those of skill in the art including: theproduction of hybrid hybridomas; disulfide exchange; chemicalcross-linking; addition of peptide linkers between two monoclonalantibodies; the introduction of two sets of immunoglobulin heavy andlight chains into a particular cell line; and so forth.

The antibodies of this invention may also be human monoclonalantibodies, for example those produced by immortalized human cells, bySCID-hu mice or other non-human animals capable of producing “human”antibodies, or by the expression of cloned human immunoglobulin genes.

In addition, it may be advantageous to couple the antibodies of thisinvention to toxins such as diphtheria, pseudomonas exotoxin, ricin Achain, gelonin, etc., or antibiotics such as penicillins, tetracyclinesand chloramphenicol.

In sum, one of skill in the art, provided with the teachings of thisinvention, has available a variety of methods which may be used to alterthe biological properties of the antibodies of this invention includingmethods which would increase or decrease the stability or half-life,immunogenicity, toxicity, affinity or yield of a given antibodymolecule, or to alter it in any other way that may render it moresuitable for a particular application.

Antibodies directed against a novel B. burgdorferi polypeptide may beused in compositions and methods for the prevention and treatment ofLyme disease as caused by infection with B. burgdorferi. For example, wedemonstrate herein that the level of B. burgdorferi in infected ticks isdecreased by allowing them to feed on the blood of animals immunizedwith OspE and OspF polypeptides. This decrease is likely due to exposureof the spirochetes in the ticks to anti-OspE and anti-OspF antibodiespresent in the blood of the immunized animals.

Accordingly, it is clear that such antibodies have utility intherapeutic and prophylactic compositions and methods directed againstLyme disease and B. burgdorferi infection.

The antibodies of this invention also have a variety of other uses. Forexample, they are useful as reagents to screen for expression of the B.burgdorferi polypeptides, either in libraries-constructed from-B.burgdorferi DNA or from other samples in which the proteins may bepresent. Moreover, by virtue of their specific binding affinities, theantibodies of this invention are also useful to purify or removepolypeptides from a given sample, to block or bind to specific epitopeson the polypeptides and to direct various molecules, such as toxins, tothe surface of B. burgdorferi.

To screen the novel B. burgdorferi polypeptides and antibodies of thisinvention for their ability to confer protection against Lyme disease ortheir ability to lessen the severity of B. burgdorferi infection, C3H/Hemice are preferred as an animal model of course, while any animal thatis susceptible to infection with B. burgdorferi may be useful, C3H/Hemice are not only susceptible to B. burgdorferi infection but are alsoafflicted with clinical symptoms of a disease that is remarkably similarto Lyme disease in humans. Thus, by administering a particularpolypeptide or antibody to C3H/He mice, one of skill in the art maydetermine without undue experimentation whether that polypeptide orantibody would be useful in the methods and compositions claimed herein.

The administration of the novel B. burgdorferi polypeptide or antibodyof this invention to the animal may be accomplished by any of themethods disclosed herein or by a variety of other standard procedures.For a detailed discussion of such techniques, see Antibodies, ALaboratory Manual, supra. Preferably, if a polvpeptide is used, it willbe administered with a pharmaceutically acceptable adjuvant, such ascomplete or incomplete Freund's adjuvant, RIBI (muramyl dipeptides) orISCOM (immunostimulating complexes). Such adjuvants may protect thepolypeptide from rapid dispersal by sequestering it in 3.0 a localdeposit, or they may contain substances that stimulate the host tosecrete factors that are chemotactic for macrophages and othercomponents of the immune system. Preferably, if a polypeptide is beingadministered, the immunization schedule will involve two or moreadministrations of the polypeptide, spread out over several weeks.

Once the novel B. burgdorferi polypeptides or antibodies of thisinvention have been determined to be effective in the screening process,they may then be used in a therapeutically effective amount inpharmaceutical compositions and methods to treat or prevent Lyme diseasewhich may occur naturally in various animals.

The pharmaceutical compositions of this invention may be in a variety ofconventional depot forms. These include, for example, solid, semi-solidand liquid dosage forms, such as tablets, pills, powders, liquidsolutions or suspensions, liposomes, capsules, suppositories, injectableand infusible solutions. The preferred form depends upon the intendedmode of administration and prophylactic application.

Such dosage forms may include pharmaceutically acceptable carriers andadjuvants which are known to those of skill in the art. These carriersand adjuvants include, for example, RIBI, ISCOM, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances, such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes such as protaminesulfate, disodium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica,-magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, and polyethylene glycol. Adjuvants fortopical or gel base forms may be selected from the group consisting ofsodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol,and wood wax alcohols.

The vaccines and compositions of this invention may also include othercomponents or be subject to other treatments during preparation toenhance their immunogenic character or to improve their tolerance inpatients.

Compositions comprising an antibody of this invention may beadministered by a variety of dosage forms and regimens similar to thoseused for other passive immunotherapies and well known to those of skillin the art. Generally, the novel B. burgdorferi polypeptides may beformulated and administered to the patient using methods andcompositions similar to those employed for other pharmaceuticallyimportant polypeptides (e.g., the vaccine against hepatitis).

Any pharmaceutically acceptable dosage route, including parenteral,intravenous, intramuscular, intralesional or subcutaneous injection, maybe used to administer the polypeptide or antibody composition. Forexample, the composition may be administered to the patient in anypharmaceutically acceptable dosage form including those which may beadministered to a patient intravenously as bolus or by continuedinfusion over a period of hours, days, weeks or months,intramuscularly—including paravertebrally andperiarticularly—subcutaneously, intracutaneously, intra-articularly,intrasynovially, intrathecally, intralesionally, periostally or by oralor topical routes. Preferably, the compositions of the invention are inthe form of a unit dose and will usually be administered to the patientintramuscularly.

The novel B. burgdorferi polypeptides or antibodies of this inventionmay be administered to the patient at one time or over a series oftreatments. The most effective mode of administration and dosage regimenwill depend upon the level of immunogenicity, the particular compositionand/or adjuvant used for treatment, the severity and course of theexpected infection, previous therapy, the patient's health status andresponse to immunization, and the judgment of the treating physician.For example, in an immunocompetent patient, the more highly immunogenicthe polypeptide, the lower the dosage and necessary number ofimmunizations. Similarly, the dosage and necessary treatment time willbe lowered if the polypeptide is administered with an adjuvant.Generally, the dosage will consist of 10 μg to 100 mg of the purifiedpolypeptide, and preferably, the dosage will consist of 100-1000 μg.Generally, the dosage for an antibody will be 0.5 mg-3.0 g.

In a preferred embodiment of this invention, the novel B. burgdorferipolypeptide is administered with an adjuvant, in order to increase itsimmunogenicity. Useful adjuvants include RIBI, and ISCOM, simple metalsalts such as aluminum hydroxide, and oil based adjuvants such as.complete and incomplete Freund's adjuvant. When an oil based-adjuvant isused, the polypeptide usually is administered in an emulsion with theadjuvant.

In yet another preferred embodiment, E. coli expressing proteinscomprising a novel B. burgdorferi polypeptide are administered orally tonon-human animals to decrease or lessen the severity of B. burgdorferiinfection. For example, a palatable regimen of bacteria expressing anovel B. burgdorferi polypeptide, alone or in the form of a fusionprotein or multimeric protein, may be administered with animal food tobe consumed by wild mice or deer, or by domestic animals. Ingestion ofsuch bacteria may induce an immune response comprising both humoral andcell-mediated components. See J. C. Sadoff et al., “Oral SalmonellaTyphimurium Vaccine Expressing Circumsporozoite Protein Protects AgainstMalaria”, Science, 240, pp. 336-38 (1988) and K. S. Kim et al.,“Immunization Of Chickens With Live Escherichia coli Expressing Eimeriaacervulina Merozoite Recombinant Antigen Induces Partial ProtectionAgainst Coccidiosis”, Inf. Immun., 57, pp. 2434-40 (1989). Moreover, thelevel of B. burgdorferi infection in ticks feeding on such animals willbe lessened or eliminated, thus inhibiting transmission to the nextanimal.

According to yet another embodiment, the antibodies of this invention aswell as the novel B. burgdorferi polypeptides of this invention, and theDNA sequences encoding them are useful as diagnostic agents fordetecting infection with B. burgdorferi, because the polypeptides arecapable of binding to antibody molecules produced in animals, includinghumans that are infected with B. burgdorferi, and the antibodies arecapable of binding to B. burgdorferi or antigens thereof.

Such diagnostic agents may be included in a kit which may also compriseinstructions for use and other appropriate reagents, preferably a meansfor detecting when the polypeptide or antibody is bound. For example,the polypeptide or antibody may be labeled with a detection means thatallows for the detection of the polypeptide when it is bound to anantibody, or for the detection of the antibody when it is bound to B.burgdorferi or an antigen thereof.

The detection means may be a fluorescent labeling agent such asfluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and thelike, an enzyme, such as horseradish peroxidase (HRP), glucose oxidaseor the like, a radioactive element such as ¹²⁵I or ⁵¹Cr that producesgamma ray emissions, or a radioactive element that emits positrons whichproduce gamma rays upon encounters with electrons present in the testsolution, such as ¹¹C, ¹⁵O, or ¹³N. Binding may also be detected byother methods, for example via avidin-biotin complexes.

The linking of the detection means is well known in the art. Forinstance, monoclonal antibody molecules produced by a hybridoma can bemetabolically labeled by incorporation of radioisotope-containing aminoacids in the culture medium, or polypeptides may be conjugated orcoupled to a detection means through activated functional groups.

The diagnostic kits of the present invention may be used to detect thepresence of a quantity of B. burgdorferi or anti-B. burgdorferiantibodies in a body fluid sample such as serum, plasma or urine. Thus,in preferred embodiments, a novel B. burgdorferi polypeptide or anantibody of the present invention is bound to a solid support typicallyby adsorption from an aqueous medium. Useful solid matrices are wellknown in the art, and include crosslinked dextran; agarose; polystyrene;polyvinylchloride; cross-linked polyacrylamide; nitrocellulose ornylon-based materials; tubes., plates or the wells of microtiter plates.The polypeptides or antibodies of the present invention may be used asdiagnostic agents in solution form or as a substantially dry powder,e.g., in lyophilized form.

Novel B. burgdorferi polypeptides and antibodies directed against thosepolypeptides provide much more specific diagnostic reagents than wholeB. burgdorferi and thus may alleviate such pitfalls as false positiveand false negative results.

One skilled in the art will realize that it may also be advantageous inthe preparation of detection reagents to utilize epitopes from other B.burgdorferi proteins, including the flagella-associated protein, andantibodies directed against such epitopes. As explained further inExample VI, infra, antibodies to OspF tend to occur late in the courseof B. burgdorferi infection while antibodies against OspE tend to appearmuch earlier. Accordingly, it may be particularly advantageous to useOspF epitopes in combination with epitopes from other B. burgdorferiproteins, such as OspE and flagellin, that elicit antibodies that occurin the early stages of Lyme disease. Diagnostic reagents containingmultiple epitopes which are reactive with antibodies appearing atdifferent times are useful to detect the presence of anti-B. burgdorferiantibodies throughout the course of infection and to diagnose Lymedisease at all stages.

The polypeptides and antibodies of the present invention, andcompositions and methods comprising them, may also be useful fordetection, prevention, and treatment of other infections caused byspirochetes which may contain surface proteins sharing amino acidsequence or conformational similarities with the novel B. burgdorferipolypeptides of the present invention. These other spirochetes includeBorrelia Hermsii and Borrelia Recurientis, Leptospira, and Treponema.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLE I Construction and Screening of a B. burgdorferi ExpressionLibrary

We had a B. burgdorferi genomic DNA expression library constructed inLambda ZAP II by Stratagene (La Jolla, Calif.) Briefly, we grew B.burgdorferi strain N40 in modified Barbour-Stoener-Kelly (BSK II) mediumat 32° C. for 7 days, harvested by centrifugation at 16,000 rpm for 30minutes, and lysed with SDS [A. G. Barbour, “Isolation and Cultivationof Lyme Disease Spirochetes”, Yale J. Biol. Med., 57, pp. 521-25(1984)]. We then isolated the genomic DNA from the spirochetes andpurified it by phenol/chloroform extraction.

To construct the library, 200 μg of DNA was randomly sheared,blunt-ended with S1 nuclease, and the EcoR1 sites were methylated withEcoR1 methylase. EcoR1 linkers were then ligated to the ends of the DNAmolecules, the DNA was digested with EcoR1and the fragments werepurified over a sucrose gradient. Fragments of 1 to 9 kb were isolatedand ligated to EcoR1 digested Lambda ZAP II arms.

We prepared E. coli SURE bacteria (Stratagene) for phage infection asfollows. We picked a single colony into LB media supplemented with 0.2%maltose and 10 nM magnesium sulfate and cultured overnight at 30° C.with vigorous shaking. We then centrifuged the cells at 2000 rpm for 10minutes and resuspended in 10 mM magnesium sulfate. The cells werefurther diluted to O.D.₆₀₀=0.5 for bacteriophage infection.

To screen the library, we used the picoBlue Immunoscreening Kit(Stratagene). We plated 10,000 plaque forming units of recombinant phageon a lawn of bacteria, induced protein expression with 10 mM IPTG andtransferred the proteins to nitrocellulose filters according to methodswell known in the art.

We prepared rabbit anti-B. burgdorferi N40 antiserum as follows. Weinjected rabbits with an inoculum of 1 ×10⁸ live B. burgdorferi N40 inPBS via the marginal ear vein and boosted with the same dosage at 14, 21and 49 days. Two weeks after the last boost, we sacrificed and bled therabbits and separated the anti-B. burgdorferi antiserum by centrifugingthe blood at 2000 rpm for 15 minutes.

To remove antibodies in the serum that would recognize E. coli and phageproteins, we absorbed the antiserum with an E. coli /phage lysate(Stratagene) as follows. We diluted the lysate 1:10 in Tris-bufferedsaline with 0.05% Tween-20 (TBST). We then incubated 0.45 μM pore sizenitrocellulose filters (Millipore, Bedford, Mass.) in the lysate for 30minutes at room temperature, removed and air dried the filters onWhatman filter paper (Whatman International Ltd., Maidstone, England),and washed 3 times (5 minutes each) with TBS. We blocked the filters byimmersing in 1% Bovine Serum Albumin (BSA) in TBS for 1 hour at roomtemperature and rinsing 3 times with TBST. We then diluted the rabbitantiserum 1:5 in TBST, incubated it with the filters with shaking for 10minutes at 37° C., and removed and discarded the filters.

After absorption, we diluted the antiserum to a final dilution of 1:200and used it to screen the nitrocellulose filters containing theexpressed proteins from the lambda ZAP library according tomanufacturer's instructions. After washing, we incubated the filterswith a 1:5000 dilution of alkaline phosphatase-conjugated goatanti-rabbit IgG antibody (Organon Teknika Corp., West Chester, Pa.), andused nitro blue tetrazolium (NBT) (Stratagene) and5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Stratagene) for colordevelopment.

Ten positive clones were originally identified. We screened each ofthese positive clones with OspA, OspB and flagellin DNA probes bySouthern blotting and identified six clones that were not reactive withany of the probes. We excised the pBluescript plasmid from one of thoseclones by infection of XL1-Blue E. coli cells and rescue with R408helper phage according to the manufacturer's instructions. We designatedthat plasmid “clone #11.”

We analyzed the protein expression of clone #11 as follows. We grew theXL1-Blue cells containing clone #11 to OD₆₀₀=0.5 (about 3 hours) andthen induced a portion of the cells with IPTG for about 2 hours. We thencentrifuged the cells at 13,000 rpm for 1 minute and resuspended thepellet in 1/10 volume of PBS with 1% Triton S-100 and 1/10 volume of 2×sample buffer. After boiling for 5 minutes, we electrophoresed thesample through a 12% SDS polyacrylamide gel and transferred overnight toa nitrocellulose filter. We blocked the filters for 1 hour with blockingsolution, incubated for 1 hour with combined mouse anti-OspE andanti-OspF anti-serum diluted 1:100, washed 3 times for 5 minutes withTBST and developed with NBT and BCIP. The mouse antiserum was preparedas set forth in Example V.

As shown in FIG. 1, the combined mouse antiserum bound to proteinshaving apparent molecular weights of 19 and 29 kDa in lysates from bothIPTG induced (Lane 1) and uninduced (Lane 2) cultures of clone #11,suggesting those proteins were expressed from their own promotersequences. Binding was absent in lysates from uninduced XL-1 Bluecontrol cells (Lane 3).

EXAMPLE II Seauence Analysis of the OspE-OspF Operon

We generated a nested set of deletions in the DNA insert of clone #11with the Erase-A-Base System (Promega, Madison, Wis.) (using SmaI togenerate the 5′ blunt end and BstXI to generated 3′ overhang). We thensequenced the subclones using the Sequenase Kit (United StatesBiochemical Corp., Cleveland, Ohio) and reconstructed the entiresequence using MacVector (International Biotechnology, Inc., New Haven,C.T). Analysis of the DNA sequence of the insert revealed that we hadisolated a novel, bicistronic B. burgdorferi operon having the sequenceset forth in SEQ ID NO: 1.

We designated the antigens encoded by the two genes in the operon asOspE and OspF. As shown in SEQ ID NO: 1, the OspE gene, at the 5′ end ofthe operon, contains a 513 nucleotide open reading frame capable ofencoding a 171-amino acid protein (SEQ ID NO: 2) with a calculatedmolecular weight of 19.2 kDa. The ATG start codon for the OspF gene islocated 27 nucleotides downstream of the TAG stop codon of the OspEgene. The OspF gene contains an open reading frame of 690 nucleotides,capable of encoding a protein of 23U amino acids with a calculatedmolecular weight of 26.1 kDa (SEQ ID NO: 3). The TAA stop codon of theOspF gene is followed by a putative stem and loop structure with dyadsymmetry.

A consensus ribosome binding site with the Shine-Dalgarno sequence islocated 10 bp gupstream of the OspE ATG start codon. Further upstream ofthis translational initiation sequence are the promoter segments knownas the “−10” region and the “−35” region, which are similar to thosefound in E. coli and other B. burgdorferi genes. (See FIG. 2 for acomparison of these regions between various B. burgdorferi genes). Anadditional ribosome binding site, consisting of a Shine-Dalgarnosequence preceded by an adenine is located 14 bp upstream of the ATGstart codon of the OspF gene. The location of these sequence elementssuggests that both the OspE and OspF genes are controlled by a singlepromoter.

Both the OspE and OspF proteins have a comparatively high content oflysine and glutamic acid (FIG. 3). Other preferred codons are leucine,isoleucine, glycine and serine (FIG. 4), with the preferred nucleotidein the wobble position being an A or a U. On the basis of amino acidcomposition, we calculated the isoelectric point of OspE and OspF as8.05 and 5.33, respectively. The hydrophilicity profiles of OspE andOspF, shown in FIG. 5 and 6, respectively, suggest that both arehydrophilic proteins.

Like OspA, OspB and OspD, the proteins encoded by the OspE and OspFgenes appear to be surface lipoproteins. As shown in FIG. 7, eachprotein begins with a basic N-terminal peptide (amino acids 1-5 of SEQID NOS: 2 and 3, respectively) followed by an amino-terminal hydrophobicdomain of about 20 amino acids that corresponds to the leader peptidefound in typical prokaryotic lipoprotein precursors [M. E. Brandt etal., supra and C. H. Wu and M. Tokunaga, “Biogenesis of Lipoproteins inBacteria”, Current Tonics in Microbiology and Immunology, 125, pp.127-157 (1986)].

The carboxyl terminus of the hydrophobic domain contains a cleavage sitepresumably recognized by a B. burgdorferi signal peptidase. In OspE, thepotential. cleavage site is located between Ala₁₉ and Cys₂₀. In OspF,the potential cleavage site is located between Ser₁₇ and Cys₁₈.

The consensus sequence of typical bacterial lipoprotein precursorsrecognized and cleaved by signal peptidase II is a leucine and acysteine separated by two, usually small, neutral, amino acids. [C. H.Wu et al., supra]. Indeed, in the OspA and OspB genes of B. burgdorferiB31, the intervening amino acids are isoleucine and alanine (OspA) orisoleucine and glycine (OspB) [S. Bergstrom et al., “Molecular Analysisof Linear Plasmid-Encoded Major Surface Proteins, OspA and OspB, of theLyme Disease Spirochaete Borrelia burgdorferi,” Mol. Microbiol., 3,479-86 (1989)]. In contrast, as shown in FIG. 7, the signal sequences ofthe B. burgdorferi N40 OspE gene (amino acids 16-20 of SEQ ID NO: 2) andOspF gene (amino acids 14-18 of SEQ ID NO: 3), like the OspC-Pko andOspD-B31 genes, contain three amino acids between the leucine andcysteine instead of two (phenylalanine, isoleucine and serine in thecase of OspC-Pko; serine, isoleucine and serine in the case ofOspD-B31). (See R. S. Fuchs et al. and S. J. Norris et al., supra).However, despite this variation in the signal sequence, OspA, OspB andOspD have been shown to be lipoproteins by the established,[³H]-palmitate labelling procedure. (See M. E. Brandt et al. and S. J.Norris et al., supra.) The leader signal sequences of OspE and OspFsuggest that these surface proteins may be processed as lipoproteins aswell. The addition of a lipid moiety at the cysteine residue could serveto anchor the proteins to the outer surface of the spirochetes (see H.C. Wu and M. Tokunaga, supra).

Finally, both OspE and OspF contain long hydrophilic domains separatedby short stretches of hydrophobic segments. However, while the first 30amino acids of OspE and OspF share a 60% homology in amino acid sequence(see FIG. 7), beyond that N-terminal region, no significant homology wasnoted.

EXAMPLE III Mapping of the OspE-OspF Operon

We mapped the OspE-OspF operon by pulsed-field electrophoresis withtotal B. burgdorferi N40 DNA using a modification of the techniquedescribed in M.S. Ferdows and A. G. Barbour, “Megabase-Sized Linear DNAin the Bacterium Borrelia burgdorferi, the Lyme Disease Agent”, Proc.Natl. Acad. Sci., 86, pp. 5969-5973 (1989). Briefly, we separated thechromosomal and plasmid DNA by loading DNA plugs containingapproximately 10⁸ B. burgdorferi N40 onto a 0.8% agarose gel. Weelectrophoresed the DNA in TBE buffer using the Chef-DRII system(Bio-Rad Laboratories, Richmond, Calif.) at 14° C. for 18 hours at 198V,with ramped pulse times from 1 to 30 sec. As shown in FIG. 8, thechromosomal band of B. burgdorferi N40 DNA migrates slightly slower thanthe 1212.5 kb marker, indicating it may be larger than previouslydescribed (1000 kb). Plasmids can be seen clearly at molecular weightsof 49 kb and lower.

After Southern blotting, we hybridized the B. burgdorferi DNA withPCR-amplified radiolabelled OspE and OspF DNA sequences. To prepare theamplified OspE DNA, we used oligonucleotide primers having the sequencesset forth in SEQ ID NO: 8 and 9. To prepare the amplified OspF DNA, weused oligonucleotide-primers having the sequences set forth in SEQ IDNO: 10 and 11. We used OspA and OspD probes as controls in the Southernblot.

As expected, the OspA and OspD probes hybridized to plasmids migratingat 49 kb and 38 kb, respectively [A. G. Barbour and C. F. Garon, “LinearPlasmids of the Bacterium Borrelia burgdorferi Have Covalently ClosedEnds”, Science, 237, pp. 409-411 (1987) and S. J. Norris et al., supra](See FIG. 9). The OspF probe bound to a plasmid which appeared tomigrate at the same molecular weight as a linear plasmid of around 45 kb(FIG. 9, lane 3) and also showed some weak binding to the 49 kb plasmidor a comigrating plasmid. The OspE probe also bound to the 45 kb plasmid(data not shown).

EXAMPLE IV Expression of OspE and OspF Polypeptides

In order to express the OspE and OspF genes, we utilized the pMX vector,which is capable of directing expression of cloned inserts asglutathione S-transferase fusion proteins [see J. Sears et al.,“Molecular Mapping of OspA-Mediated Immunity to Lyme Borreliosis”, J.Immunol., 147, pp. 1995-2000 (1991)]. We first used PCR to amplify OspEand OspF genes lacking the sequences encoding the hydrophobic leaderpeptides. We chose to delete those sequences to ensure that OspE andOspF would be expressed as soluble fusion proteins rather than aslipoproteins, which would be anchored to the cell membrane.

To amplify the OspE gene, we selected the primers shown in SEQ ID NO: 8and 9. These primers allow amplification of a DNA sequence encodingamino acids 21-171 of SEQ ID NO: 2, flanked by a BamH1 site on the 5′end and an Xho I site on the 3′ end. The primers we used to amplify theOspF gene, shown in SEQ ID NO: 10 and 11, result in amplification of aDNA sequence encoding amino acids 19-230 of SEQ ID NO: 3, flanked by anEcoR1 site on the 5′ end and an Xho I site on the 3′end. Theamplification was conducted for 30 cycles with initial templatedenaturation at 94° C. for 1 minute, annealing at 40′ C. for 2 minutesand extension at 72° C. for 3 minutes.

We then ligated the amplified sequences to appropriately digested pMXvector and transformed DH5 αE. coli according to methods well known tothose of skill in the art. After selecting subclones containing thedesired inserts, we cultured the cells and induced expression of theOspE and OspF genes as glutathione S-transferase fusion proteins.

We purified the glutathione S-transferase OspE and OspF fusion proteins(GT-OspE and GT-OspF, respectively) from cell lysates using aglutathione-Sepharose 4B column (Pharmacia) according to themanufacturer's instructions. In addition, we purified the OspE and OspFproteins without the glutathione S-transferase sequences as follows. Weloaded the OspE and OspF glutathione S-transferase fusion proteins overthe glutathione-Sepharose 4B column, added 25 units of thrombin andincubated overnight at room temperature. We then eluted the proteinswith 50 mM Tris-CaCl₂-NaCl, treated the eluent with anti-thrombin beadsfor 1.5 to 2 hours and centrifuged at 13,000 rpm. The purifiedrecombinant OspE (Δ20-OspE) and OspF (Δ18-OspF) polypeptides obtainedfrom this procedure are shown in lanes 2 and 3, of FIG. 10,respectively.

EXAMPLE V Preparation of Anti-OspE and Anti-OspF Antibodies

To determine whether OspE or OspF polypeptides were capable of elicitingan immune response, we immunized C3H/HeJ mice (Jackson Laboratory, BarHarbor, Me.) subcutaneously with 10 micrograms of-either GT-OspE,GT-OspF, Δ20-OspE or Δ18-OspF in complete Freund's adjuvant and boostedwith the same amount in incomplete Freund's adjuvant at 14 and 28 days.Control mice were immunized in the same manner with either recombinantglutathione S-transferase or bovine serum albumin (BSA). We alsoimmunized white New Zealand rabbits (Millbrook, Amherst, Mass.) in asimilar fashion with 50 micrograms of either Δ20-OspE or Δ18-OspF toobtain antisera for immunofluorescence studies.

Ten days after the last boost, we collected sera from the immunizedanimals and used it to hybridize to Western blots of SDS-PAGE gels of B.burgdorferi N40 extract or various of the recombinant polypeptides. Wedetected binding with a 1:5200 dilution of alkaline phosphatase-labeledgoat anti-mouse immunoglobulin G and developed with nitrobluetetrazolium and 5-bromo-4-chloro-indolyl phosphate. Alternatively, weused the ECL kit (Amersham, Arlington Heights, Ill.) in which thesecondary antibody, horseradish peroxidase-labeled goat anti-mouse IgG,can be detected at a dilution of 1:4000.

All of the OspE and OspF immunogens elicited antibodies in mice thatwere detectable by immunoblotting at a dilution of 1:5000. Similarly,Δ20-OspE and Δ18-OspF elicited antibodies in the immunized rabbits thatwere detectable by immunoblotting at a dilution of 1:1000. We note thatthe relatively low antibody titers suggest that the recombinant OspE andOspF molecules used are not particularly immunogenic. For example,immunization with GT-OspA elicited a titer in rabbits of 1:10,000,000.

We also used the antiserum from the immunized animals to confirm theidentity of the cloned polypeptides. As shown in FIG. 11 (lane 2),antibodies from mice immunized with Δ20-OspE bound to a protein in B.burgdorferi extract which migrated at approximately 19 kDa. This sizeapproximates the predicted size of the OspE protein of SEQ ID NO: 2 andthus could represent the processed, lipidated form of the OspE protein.

Mice immunized with Δ18-OspF detected two B. burgdorferi proteins (FIG.11, lane 3). One migrated at approximately 29 kDa—the approximate sizefor a processed, lipidated OspF protein. The other protein migrated atapproximately 36 kDa. Because the 36 kDa protein is immunologicallycross-reactive with antibodies directed against an OspF polypeptide ofthis invention, it is, thus, also an OspF polypeptide of this invention.

The 36 kDa OspF polypeptide may be isolated by a variety of methodsavailable to one of skill in the art. For example, anti-OspF antiserumcould be used to screen the B. burgdorferi expression libraryconstructed in Example I for clones capable of expressing that protein.Alternatively, an expression library could be constructed in whichsmaller fragments of B. burgdorferi DNA are cloned in frame into anexpression vector from which they would be expressed as glutathioneS-transferase rusion proteins, such as pGEX-2T, pMX, or pGEMEX. Such alibrary would have a high likelihood of expressing the sequence as afusion protein, even if it is normally linked to a promoter that is nottranscriptionally active in E. coli.

Alternatively, the protein may be purified by immunoprecipitation,segments of the amino acid sequence determined, and oligonucleotidessynthesized, which may then be used to screen-a genomic B. burgdorferilibrary

We also used the rabbit anti-OspE and anti-OspF antisera inimmunofluorescence studies to verify that OspE and OspF are expressed onthe outer surface of the B. burgdorferi spirochete. Rabbit antiseradirected against both Δ20-OspE and Δ18-OspF stained fixed B. burgdorferiN40, although at approximately only half the intensity as that achievedwith antibodies directed against whole B. burgdorferi or recombinantOspA. FIG. 12(A) shows the staining pattern of spirochetes fixed withparaformaldehyde-and stained in suspension with rabbit anti-OspE seradiluted 1:100. FIG. 12(B) shows the staining pattern when anti-OspBdiluted 1:100 is used. The staining pattern indicates that both the OspEand OspF proteins are exposed on the outer membrane of the spirochetes.

EXAMPLE VI Characterization of the Immune Response to OspE and OspF

To further characterize the immune response to OspE and OspF during thecourse of infection, we infected normal mice intradermally with 10⁴ B.burgdorferi N40 and collected sera at day 30 and day 90 after infection.We then used this sera on Western blots of purified Δ20-OspE andΔ18-OspF polypeptides.

FIG. 13(a) shows that sera taken from mice 30 days after infection boundto Δ20-OspE (lane 1) but not to Δ18-OspF (lane 2). FIG. 13(b) shows thatby 90 days after infection, anti-OspF antibodies were detectable,although at lower levels than those directed against OspE.

We also characterized the human immune response to the OspE and OspFproteins. We obtained sera from 28 patients with early stage Lymedisease (defined as patients having erythema migrans on the day ofdiagnosis and skin lesions for less than 1 week) and from 19 patientswith late stage Lyme-disease (defined as patients having erythemamigrans for at least six months).

As shown in Table I, below, 11% of the early stage Lyme disease patientshad antibodies to OspE and 14% had antibodies to OspF. However, in thelate stage patients, while only 15% had antibodies to OspE, 58% hadantibodies to OspF.

TABLE I Number (%) of patients Stage of No. of with antibodies todisease patients B. burgdorferi OspE OspF Early 28 26(93) 3(11)  4(14)Late 19 19(100) 3(15) 11(58)

A representative immunoblot of the Δ20-OspE and Δ18-OspF polypeptidesprobed with human sera from a late-stage Lyme disease patient is shownin FIG. 13(c).

EXAMPLE VII Ability of OspE and OspF to Protect Against B. burgdorferiInfection

To determine whether the OspE or OspF polypeptides were able to elicitan immune response that would be effective to protect against B.burgdorferi infection, we actively immunized C3H/He mice with thevarious OspE and OspF polypeptides described above, and then attemptedto infect the immunized mice with B. burgdorferi N40.

We grew a low passage isolate of B. burgdorferi N40, with demonstratedinfectivity and pathogenicity, to log phase in BSK II medium and countedwith a hemocytometer under dark-field microscopy. We then challenged theactively immunized mice approximately 14 days after the last boost withintradermal inoculations of 10² or 10⁴ spirochetes and sacrificedfourteen days after infection. We then cultured selected mouse tissuesin BSK II medium to assay for spirochetes, and examined joints andhearts for inflammation.

As shown below in Table II, when mice immunized with either GT-OspF orΔ8-OspF were challenged intradermally with 10² B. burgdorferi, theyexhibited a low frequency of infection and disease in comparison tocontrol mice (P<0.05 by χ² test), indicating that OspF is capable ofproviding protection from B. burgdorferi infection. This protectiveeffect was no longer apparent when the dose of spirochetes was increasedto 10⁴.

However, that dose is likely to be considerably greater than thatdelivered by tick bite. In contrast, the mice immunized with GT-OspE orΔ20-OspE did not appear to be significantly protected from subsequentinfection, regardless of the dose of B. burgdorferi administered.

TABLE II Immununization Challenge (Active) Culture^(a) Disease^(b)Infection^(c) 10⁴ N40 OspE 3/5 3/5 4/5 OspF 3/5 2/5 3/5 Control 3/5 2/53/5 10² N40 OspE 4/9 2/9 4/9 OspF 1/9 0/9 1/9 Control 5/9 2/9 5/9 ^(a)Amouse was considered positive if spirochetes could be cultured fromblood, spleen or bladder. ^(b)Histological examination of joints andhearts. ^(c)An animal was “infection” positive if it had a positiveculture, evidence of disease, or both.

EXAMPLE VIII Protection Against Tick-mediated Transmission

We also determined whether the protection conferred by immunization withOspF extended to tick-mediated transmission of the spirochete. Weobtained spirochete-free Ixodes dammini ticks from the Harvard School ofPublic Health, which maintains a laboratory colony derived from anIpswich, MA population. We infected the ticks (at the larval stage) byallowing them to feed to repletion on outbred CD-1 mice that had beenpreviously infected (three weeks prior to serving as hosts) byintradermal inoculation of 10³ B. burgdorferi N40 spirochetes. Uponrepletion, we collected engorged larvae, pooled them in groups of100-200, and permitted them to molt to the nymphal stage at 21° C. and95% relative humidity. We determined the prevalence of infection in eachpool by immunofluorescence of a representative sample (10 ticks) threeweeks after molting. We used only those pools having an infectionprevalence of greater than 70% for challenge experiments.

We immunized mice with GT-OspE or Δ20-OspE and GT-OspF or Δ18-OspF orwith BSA as a control, as described in Example V. Two weeks after thelast boost, we placed 3 to 5 nymphal ticks on each mouse, allowed themto feed or repletion and then allowed them to detach naturaly overwater. Two weeks later we sacrificed the mice in order to culture thetissues for spirochetes and examine the organs, as described above.

As shown below in Table III, the protective effect of immunization withOspF was again evident. In contrast, the frequency of B. burgdorferiinfection in mice immunized with OspE appeared to be the same as in thecontrol mice that had not been immunized.

TABLE III Immunization Culture^(a) Disease^(b) Infection^(c) OspE 6/125/12 7/12 OspF 2/12 2/12 2/12 Control 6/12 7/12 7/12 ^(a)A mouse wasconsidered positive if spirochetes could be cultured from blood, spleenor bladder. ^(b)Histological examination of joints and hearts. ^(c)Ananimal was “infection” positive if it had a positive culture, evidenceof disease, or both.

EXAMPLE IX Decrease in Spirochete Load in Ticks Feeding on ImmunizedAnimals

Previous studies have shown that immunization of mice with recombinantOspA can eliminate the spirochetes from ticks feeding on the immunizedanimals [E. Fikrig et al., “Elimination of Borrelia burgdorferi fromvector ticks feeding on OspA-immunized mice”, Proc. Natl. Acad. Sci.,89, pp. 5418-5421 (1992)]. Thus, we sought to determine if spirocheteswould also be killed when infected ticks fed on animals immunized withOspE or OspF.

We placed five Ixodes dammini ticks, infected as described in ExampleVIII, on each of 12 control mice, 12 mice immunized with Δ20-OspE or 12mice immunized with Δ18-OspF. After feeding to repletion, the ticks wereallowed to naturally detached over water. Only a portion of the tickswere recovered from each group, the remainder apparently having beeningested by the mice. We homogenized individual ticks in 100 μl of PBSand spotted 10 μl aliquots on each of three slides. The slides wereallowed to air-dry, fixed in cold acetone for 10 minutes, and assayed bydirect or indirect immunofluorescence.

For the direct immunofluorescence assay, we stained with FITC-conjugatedrabbit anti-B. burgdorferi serum at a dilution of 1:100. In the indirectimmunofluorescence assay, we stained with the anti-OspA monoclonalantibody H5332 diluted 1:8 as a primary antibody, and FITC-conjugatedgoat anti-mouse IgG at a dilution of 1:100 as a secondary antibody. Wequantified the spirochetes by counting the number of fluorescing cellsin approximately 20 fields per slide.

As shown below in Table IV, while 85% of the ticks recovered from thecontrol mice remained infected with spirochetes, only about 58% of theticks that had fed on Δ20-OspE-immunized mice (P<0.05 by c² test), and54% of the ticks that had fed on Δ18-OspF-immunized mice (P<0.01)remained infected. Moreover, the infected ticks that were recovered fromthe OspE- and OspF-immunized mice showed a much lower spirochete loadthan those that had fed on normal control mice. For example, whileapproximately 65% of the ticks that fed on control mice were found tocarry more than 100 spirochetes/tick, that level of infection wasmaintained in only 17% of the ticks that had fed on OspE-immunized mice,and none of the ticks that had fed on OspF-immunized mice. Accordingly,while killing of spirochetes and protection against Lyme disease may notbe absolute, we have shown that immunization with OspE polypeptides,and, to an even greater extent, OspF polypeptides, is able to prevent orlessen the severity of B. burgdorferi infection by effectivelydecreasing the spirochete load in infecting ticks.

TABLE IV % infected ticks # Ticks No. (%) No. of spirochetesImmunization examined infected (1-3) (3-50) (50-100) (≦500) (>500) OspE12  7 (58) 8 25  8 17 0 OspF 24 13 (54) 8 33 13  0 0 Control 20 17 (85)0  0 20 10 55 

EXAMPLE X Passive Immunization of Mice With Anti-OspF Antiserum

Because immunization with OspF polypeptides was able to conferprotection against Lyme disease and B. burgdorferi infection, we soughtto determine if passive immunization of mice with antiserum from OspFimmunized animals would also confer protection. We passively immunizedmice with 0.2 ml of sera from rabbits immunized with Δ18-OspF. We thenchallenged the passively immunized mice with 10² B. burgdorferi N40 atone day after the immunization. Surprisingly, while immunization withOspF polypeptides is effective to prevent or lessen the severity of B.burgdorferi infection, the data shown below in Table V demonstrate thatpassive immunization with anti-OspF antiserum does not protect naiveanimals from subsequent infection.

TABLE V Passive Challenge Immunization Culture^(a) Disease^(b)Infection^(c) 10²N40 anti-OspF 8/15 3/15 8/15 antiserum control 7/152/15 7/15 ^(a)A mouse was considered positive if spirochetes could becultured from blood, spleen or bladder. ^(b)Histological examination orjoints and hearts. ^(c)An animal was “infection” positive if it had apositive culture, evidence of disease, or both.

EXAMPLE XI Restriction Analysis of Additional Clones Comprising Novel B.burgdorferi Polypeptides

As discussed in Example I, we had originally identified six clones fromthe B. burgdorferi expression library that did not hybridize with DNAprobes to OspA, OspB or flagellin. In addition to clone #11, whichcontained the OspE-F operon, we had designated the remaining clones #4,#5, #7, #8, and #10. Restriction mapping of the clones indicated thatclone #4 had a DNA insert of approximately 4.7 kb, clone #7 had a DNAinsert of approximately 4.8 kb, clone #8 had a DNA insert ofapproximately 4.3 kb, and clone #10 had a DNA insert cf approximately2.6 kb. We have not yet determined precisely the size of the DNA insertin clone #5.

EXAMPLE XI Analysis of the B. burgdorferi T5 Protein

We excised the pBluescript plasmid from clone #10 as described inExample I. We then sequenced the DNA insert as described in Example II,after generating nested deletions using the Erase-A-Base System (withXhoI to generate the 5′ blunt end and KpnI to generate the 3′ overhang.)

The nucleotide sequence of the B. burgdorferi gene contained withinclone #10 is shown in SEQ ID NO: 6.

The gene contains a 582-bp open reading frame capable of encoding a 194amino acid protein (SEQ ID NO: 7) with a predicted molecular weight of21.8 kd. We designated this protein the “T5” protein. As for the OspEand OspF genes, the gene encoding T5 contains a ribosomal binding sitesimilar to the Shine-Dalgarno sequence and −10 and −35 promoter regionssimilar to those found in E. coli and other B. burgdorferi genes.

The hydrophilicity profile (FIG. 14) and deduced amino acid sequence ofT5 (SEQ ID NO: 7) reveal a leader peptide similar to those found intypical prokaryotic lipoprotein precursors. The leader signal sequencebegins with a short positively charged peptide at the amino terminus,followed by a hydrophobic domain of 16 amino acids. At the carboxyterminus of the hydrophobic core is a signal peptidase II cleavage sitethat is similar to that of the OspE and OspF genes (amino acid 18-22 ofSEQ ID NO: 7) The lipoprotein processing site is followed by a stretchof about 30 hydrophilic amino acids. Beyond that domain, thehydrophilicity plot shows mixed intervals of hydrophobic and hydrophilicregions.

EXAMPLE XII Expression and Purification of T5

We amplified the T5 DNA sequence lacking the portion coding for theleader peptide which contains the lipidation signal sequence, cloned theinsert into pMX, expressed the protein as a glutathione S-transferasefusion protein, and cleaved the B. burgdorferi T5 protein sequences asdescribed for the OspE and OspF proteins in Example IV. We thenelectrophoresed the cleaved protein on an SDS-PAGE gel. As shown in FIG.15, the cleaved protein migrated at an apparent molecular weight of 22kDa.

EXAMPLE XIII Chromosomal Mapping of the T5 Gene

We performed pulsed-field electrophoresis as described in Example III,blotted the gel, and hybridized with various B. burgdorferi probes. Asshown in FIG. 16 (lane 3), the T5 probe hybridized to the B. burgdorferiband corresponding to the linear chromosome. Accordingly, unlike theother outer surface proteins identified to date, it appears that the T5protein is not encoded by a plasmid of B. burgdorferi. This couldsuggest that the T5 protein may be more conserved among various B.burgdorferi isolates than the plasmid-encoded outer surface proteins.

Given the DNA sequence encoding the T5 protein, one of skill in the artcan easily determine the degree of variability of this protein acrossvarious B. burgdorferi strains, either by producing antibodies againstthe protein and determining the extent of cross-reactivity, or by usingthe DNA as a probe for hybridization to the DNA of different B.burgdorferi isolates. The various isolates would then be sequenced todetermine the precise differences.

EXAMPLE XIV Sequence Analysis of the B. burgdorferi S1 Protein

We also determined the sequence of the B. burgdorferi DNA containedwithin clone #8. That DNA sequence is shown in SEQ ID NO: 4. The DNAcontains an open reading frame of 1251 base pairs, capable of encoding aprotein of 417 amino acids (SEQ ID NO: 5) with a predicted molecularweight of 48.9 kDa. We designated this protein the “S1” protein. As forOspE and OspF the gene encoding S1 contains a ribosomal binding sitesimilar to the Shine-Dalgarno sequence and −10 and −35 promoter regionssimilar to those found in E. coli and other B. burgdorferi genes.

The hydrophilicity profile (data not shown) and deduced amino acidsequence of S1 reveal a leader peptide similar to those found in typicalprokaryotic lipoprotein precursors. The leader signal sequence consistsof a hydrophobic domain of 18 amino acids. At the carboxyl terminus ofthe hydrophobic core is a signal peptidase II cleavage site that issimilar to that of the OspE and OspF genes (amino acid 14-18 of SEQ IDNO: 5).

EXAMPLE XV Analysis of Additional Novel B. burgdorferi Polypeptides

We excise the pBluescript plasmids from clones #4, #5 and #7 asdescribed above. We then sequence the clones and insert the sequences,in whole or in part, into expression vectors in order to express novelB. burgdorferi polypeptides. We then use each of the novel B.burgdorferi polypeptides of this invention to immunize rabbits or C3H/Hemice and demonstrate the ability of the polypeptides to prevent orlessen the severity, for some period of time, of B. burgdorferiinfection.

EXAMPLE XVI Determination of Protective Epitopes

We construct recombinant genes which will express fragments of the novelB. burgdorferi polypeptides in order to determine which fragmentscontain protective epitopes. First, we produce overlapping 200-300 bpfragments which encompass the entire nucleotide sequence of each of thegenes, either by restriction enzyme digestion, or by amplification ofspecific sequences of using PCR and oligonucleotide primers containingrestriction endonuclease recognition'sequences, as described supra. Wethen clone these fragments into an appropriate expression vector,preferably a vector from which the fragments will be expressed as fusionproteins, in order to facilitate purification and increase stability.For example, the gene fragments could be cloned into pGEMEX (Promega,Madison, Wis.) and expressed as T7 gene 10 fusion proteins. Suchproteins would be insoluble and thus easily purified by recovery of theinsoluble pellet fraction followed by solubilization in denaturants suchas urea. Alternatively, the fragments could be expressed as glutathioneS-transferase fusion proteins as described above. We then transformappropriate host cells and induce expression of the fragments.

One way to identify fragments that contain protective B-cell epitopes isto use the individual purified fragments to immunize C3H/HeJ mice, asdescribed above. After challenge of the nice with B. burgdorferi, wedetermine the presence of infection by blood and spleen cultures and byhistopathologic examination of the joints and heart.

Another technique to identify protective epitopes is to use the variousfragments to immunize mice, allow ticks infected with B. burgdorferi tofeed on the mice, and then determine, as set forth in Example VIII,whether the immune response elicited by the fragments is sufficient tocause a decrease in the level of B. burgdorferi in the ticks. Anyepitopes which elicit such a response, even if they are not sufficientby themselves to confer protection against subsequent infection with B.burgdorferi, may be useful in a multicomponent vaccine.

Once we have localized various epitopes to particular regions of thefusion proteins, we conduct further analyses using short syntheticpeptides of 5-35 amino acids. The use of synthetic peptides allows us tofurther define each epitope, while eliminating any variables contributedby the non-B. burgdorferi portion of the fusion protein.

EXAMPLE XVII Preparation of a Multicomponent Vaccine

We determine which of the protective epitopes is able to elicitantibodies that will protect against subsequent infection with strainsof B. burgdorferi other than the strain from which the Osp gene wascloned. We then design a vaccine around those epitopes. If none of theprotective epitopes is able to confer protection against infection withother strains of B. burgdorferi, it may be particularly advantageous toisolate the corresponding novel B. burgdorferi polypeptides from thosestrains. A multicomponent vaccine may then be constructed that comprisesmultiple epitopes from several different B. burgdorferi isolates. Such avaccine will, thus, elicit antibodies that will confer protectionagainst a variety of different strains.

EXAMPLE XVIII Identification of T Cell Epitopes

Stimulation in animals of a humoral immune response containing hightiter neutralizing antibodies will be facilitated by antigens containingboth T cell and B cell epitopes. To identify those polypeptidescontaining T cell epitopes, we infect C3H/HeJ mice with B. burgdorferistrain N40 in complete Freund's adjuvant, as described supra. Ten daysafter priming, we harvest the lymph nodes and generate in vitro T celllines. These T cell lines are then cloned using limiting dilution andsoft agar techniques. We use these T cell clones to determine whichpolypeptides contain T cell epitopes. The T cell clones are stimulatedwith the various polypeptides and syngeneic antigen presenting cells.Exposure of the T cell clones to the polypeptides that contain T cellepitopes in the presence of antigen presenting cells causes the T cellsto proliferate, which we measure by ³H-Thymidine incorporation. We alsomeasure lymphokine production by the stimulated T cell clones bystandard methods.

To determine T cell epitopes of the polypeptides recognized by human Tcells, we isolate T cell clones from B. burgdorferi-infected patients ofmultiple HLA types. T cell epitopes are identified by stimulating theclones with the various polypeptides and measuring ³H-Thymidineincorporation. The various T cell epitopes are then correlated withClass II HLA antigens such as DR, DP, and DQ. The correlation isperformed by utilization of B lymphoblastoid cell lines expressingvarious HLA genes. When a given T cell clone is mixed with theappropriate B lymphoblastoid cell line and a novel B. burgdorferipolypeptide, the B cell will be able to present the polypeptide to the Tcell. Proliferation is then measured by ³H-Thymidine incorporation.

Alternatively, T cell epitopes may be identified by adoptive transfer ofT cells from mice immunized with various of the novel B. burgdorferipolypeptides of this invention to naive mice, according to methods wellknown to those of skill in the art. [See, for example, M. S. DeSouza etal., “Long-Term Study of Cell-Mediated Responses to Borrelia burgdorferiin the Laboratory Mouse”, Infect. Immun., 61, pp. 1814-22 (1993)].

We then synthesize a multicomponent vaccine based on different T cellepitopes. Such a vaccine is useful to elicit T cell responses in a broadspectrum of patients with different HLA types.

We also identify stimulating T cell epitopes in other immunogenic B.burgdorferi polypeptides or in non-B. burgdorferi polypeptides anddesign multicomponent vaccines based on these epitopes in conjunctionwith B cell and T cell epitopes from the novel B. burgdorferipolypeptides of this invention.

EXAMPLE XIX Construction of Fusion Proteins Comprising T and B cellEpitopes

After identifying T cell epitopes of the novel B. burgdorferipolypeptides, we construct recombinant proteins comprising theseepitopes as well as the B cell epitopes recognized by neutralizingantibodies. These fusion proteins, by virtue of containing both T celland B cell epitopes, permit antigen presentation to T cells by B cellsexpressing surface immunoglobulin. These-T cells in turn stimulate Bcells that express surface immunoglobin, leading to the production ofhigh titer neutralizing antibodies.

We also construct fusion proteins from the novel B. burgdorferipolypeptides by linking regions of the polypeptides determined tocontain B cell epitopes to strong T cell epitopes of other antigens. Wesynthesize an oligonucleotide homologous to amino acids 120 to 140 ofthe Hepatitis B virus core antigen. This region of the core antigen hasbeen shown to contain a strong T cell epitope [D. R. Millich, et al.,supra]. The oligonucleotide is then ligated to the 5′ and 3′ ends ofsegments of DNA encoding the B cell epitopes recognized by neutralizingantibodies, as in Example XI. The recombinant DNA molecules are thenused to express a fusion protein comprising a B cell epitope from thenovel B. burgdorferi polypeptide and a T cell epitope from the coreantigen, thus enhancing the immunogenicity of the polypeptide.

We also construct fusion proteins comprising epitopes of the novel B.burgdorferi polypeptides as well as epitopes of the tetanus toxoidprotein.

We also construct a plasmid containing the B cell epitopes of various ofthe novel B. burgdorferi polypeptides incorporated into the flagellinprotein of Salmonella. Bacterial flagellin are potent stimulators ofcellular and humoral responses, and can be used as vectors forprotective antigens [S. M. C. Newton, C. Jacob, B. Stocker, “ImmuneResponse To Cholera Toxin Epitope Inserted In Salmonella Flagellin”,Science, 244, pp. 70-72 (1989)]. We cleave the cloned H 1-d flagellingene of Salmonella muenchens at a unique Eco RV site in thehypervariable region. We then insert blunt ended DNAs encodingprotective B cell epitopes of the polypeptides using T4 DNA ligase. Therecombinant plasmids are then used to transform non-flagellate strainsof Salmonella for use as a vaccine. Mice are immunized with live andformalin killed bacteria and assayed for antibody production. Inaddition spleen cells are tested for proliferative cellular responses tothe peptide of interest. Finally the mice immunized with this agent arechallenged with B. burgdorferi as described supra.

We also construct fusion proteins comprising B cell epitopes from one ofthe novel B. burgdorferi polypeptides and T cell epitopes from adifferent novel B. burgdorferi polypeptide or other immunogenic B.burgdorferi polypeptides. Additionally, we construct fusion proteinscomprising T cell epitopes from novel B. burgdorferi polypeptides and Bcell epitopes from a novel B. burgdorferi polypeptide and/or otherimmunogenic B. burgdorferi polypeptides. Construction of these fusionproteins is accomplished by recombinant DNA techniques well known tothose of skill in the art. Fusion proteins and antibodies directedagainst them, are used in methods and composition to detect, treat, andprevent Lyme disease as caused by infection with B. burgdorferi.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments which utilize the processes and products of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims, rather than by the specificembodiments which have been presented by way of example.

11 1498 base pairs nucleic acid double linear DNA (genomic) NO NO CDS129..644 CDS 672..1364 1 GTTGGTTAAA ATTACATTTG CGTTTTGTTA ATATGTAACAGCTGAATGTA ACAAAATTAT 60 ATATTTAAAT CTTTGAAAAA TTGCAATTAT TATGTATTGTGGTAAGATTA GGACTTATGG 120 AGTAACTT ATG AAT AAG AAA ATG AAA ATG TTT ATTGTT TAT GCT GTT TTT 170 Met Asn Lys Lys Met Lys Met Phe Ile Val Tyr AlaVal Phe 1 5 10 ATA CTT ATA GGT GCT TGC AAG ATT CAT ACT TCA TAT GAT GAGCAA AGT 218 Ile Leu Ile Gly Ala Cys Lys Ile His Thr Ser Tyr Asp Glu GlnSer 15 20 25 30 AGT GGT GAG TCA AAA GTT AAA AAA ATA GAA TTC TCT AAA TTTACT GTA 266 Ser Gly Glu Ser Lys Val Lys Lys Ile Glu Phe Ser Lys Phe ThrVal 35 40 45 AAA ATT AAA AAT AAA GAT AAA AGT GGT AAC TGG ACA GAC TTA GGAGAT 314 Lys Ile Lys Asn Lys Asp Lys Ser Gly Asn Trp Thr Asp Leu Gly Asp50 55 60 TTA GTT GTA AGA AAA GAA GAA AAT GGT ATT GAT ACG GGT TTA AAC GCT362 Leu Val Val Arg Lys Glu Glu Asn Gly Ile Asp Thr Gly Leu Asn Ala 6570 75 GGG GGA CAT TCG GCT ACA TTC TTT TCA TTA GAA GAG GAA GTA GTT AAT410 Gly Gly His Ser Ala Thr Phe Phe Ser Leu Glu Glu Glu Val Val Asn 8085 90 AAC TTT GTA AAA GTA ATG ACT GAA GGC GGA TCA TTT AAA ACT AGT TTG458 Asn Phe Val Lys Val Met Thr Glu Gly Gly Ser Phe Lys Thr Ser Leu 95100 105 110 TAT TAT GGA TAT AAG GAA GAA CAA AGT GTT ATA AAT GGT ATC CAAAAT 506 Tyr Tyr Gly Tyr Lys Glu Glu Gln Ser Val Ile Asn Gly Ile Gln Asn115 120 125 AAA GAG ATA ATA ACA AAG ATA GAA AAA ATT GAT GGA ACT GAA TATATT 554 Lys Glu Ile Ile Thr Lys Ile Glu Lys Ile Asp Gly Thr Glu Tyr Ile130 135 140 ACA TTT TCA GGA GAT AAA ATT AAG AAT TCA GGA GAT AAA GTT GCTGAA 602 Thr Phe Ser Gly Asp Lys Ile Lys Asn Ser Gly Asp Lys Val Ala Glu145 150 155 TAT GCA ATA TCA CTA GAA GAG CTT AAG AAG AAT TTA AAATAGAAGTTGG 651 Tyr Ala Ile Ser Leu Glu Glu Leu Lys Lys Asn Leu Lys 160165 170 AAGTATAGGA GAAAGCTTAT ATG AAT AAA AAA ATG TTT ATT ATT TGT GCT701 Met Asn Lys Lys Met Phe Ile Ile Cys Ala 1 5 10 ATT TTT GCG CTG ATAGTT TCT TGC AAG AAT TAT ACA ACT AGC AAA GAT 749 Ile Phe Ala Leu Ile ValSer Cys Lys Asn Tyr Thr Thr Ser Lys Asp 15 20 25 TTA GAA GGG TCA GTG CAAGAT TTA GAA AGT TCA GAA CAA AAT GCA AAA 797 Leu Glu Gly Ser Val Gln AspLeu Glu Ser Ser Glu Gln Asn Ala Lys 30 35 40 AAA ACA GAA CAA GAG ATA AAAAAA CAA GTT GAA GGA TTT TTA GAA ATT 845 Lys Thr Glu Gln Glu Ile Lys LysGln Val Glu Gly Phe Leu Glu Ile 45 50 55 CTA GAG ACA AAA GAT TTG AAT ACATTG AAT ACA AAA GAT ATA AAA GAG 893 Leu Glu Thr Lys Asp Leu Asn Thr LeuAsn Thr Lys Asp Ile Lys Glu 60 65 70 ATT GAA AAA CAA ATT CAA GAA TTA AAGGAC ACA ATA AAT AAA TTA GAG 941 Ile Glu Lys Gln Ile Gln Glu Leu Lys AspThr Ile Asn Lys Leu Glu 75 80 85 90 GCT AAA AAA ACT TCT CTT AAA ACA TATTCT GAG TAT GAA GAA CAA ATA 989 Ala Lys Lys Thr Ser Leu Lys Thr Tyr SerGlu Tyr Glu Glu Gln Ile 95 100 105 AAA AAA ATA AAA GAA AAA TTA AAA GATAAG AAA GAA CTT GAA GAT AAA 1037 Lys Lys Ile Lys Glu Lys Leu Lys Asp LysLys Glu Leu Glu Asp Lys 110 115 120 TTA AAG GAA CTT GAA GAG AGC TTA AAAAAG AAA AAA GAG GAG AGA AAA 1085 Leu Lys Glu Leu Glu Glu Ser Leu Lys LysLys Lys Glu Glu Arg Lys 125 130 135 AAA GCT TTA GAA GAT GCT AAG AAG AAATTT GAA GAG TTT AAA GGA CAA 1133 Lys Ala Leu Glu Asp Ala Lys Lys Lys PheGlu Glu Phe Lys Gly Gln 140 145 150 GTT GGA TCC GCA ACC GGA CAA ACT CAAGGG CAG AGA GCT GGA AAT CAG 1181 Val Gly Ser Ala Thr Gly Gln Thr Gln GlyGln Arg Ala Gly Asn Gln 155 160 165 170 GGG CAG GTT GGA CAA CAA GCT TGGAAG TGT GCT AAT AGT TTG GGG TTG 1229 Gly Gln Val Gly Gln Gln Ala Trp LysCys Ala Asn Ser Leu Gly Leu 175 180 185 GGT GTA AGT TAT TCT AGT AGT ACTGGT ACT GAT AGC AAT GAA TTG GCA 1277 Gly Val Ser Tyr Ser Ser Ser Thr GlyThr Asp Ser Asn Glu Leu Ala 190 195 200 AAC AAA GTT ATA GAT GAT TCA ATTAAA AAG ATT GAT GAA GAG CTT AAA 1325 Asn Lys Val Ile Asp Asp Ser Ile LysLys Ile Asp Glu Glu Leu Lys 205 210 215 AAT ACT ATA GAA AAT AAT GGA GAAGTC AAA AAA GAA TAAAGAAATG 1371 Asn Thr Ile Glu Asn Asn Gly Glu Val LysLys Glu 220 225 230 GTTTTTAAAA GTATAAATTA CGAAAAACAA GACTAATAACCAGTCTTGTT TTTTTATTTA 1431 AGCCATACTT TTATGAAGTG AAAATGCCAA AAACTATTGTTAAAAATGTT GTTTATTTAT 1491 ACATTCT 1498 171 amino acids amino acidlinear protein 2 Met Asn Lys Lys Met Lys Met Phe Ile Val Tyr Ala Val PheIle Leu 1 5 10 15 Ile Gly Ala Cys Lys Ile His Thr Ser Tyr Asp Glu GlnSer Ser Gly 20 25 30 Glu Ser Lys Val Lys Lys Ile Glu Phe Ser Lys Phe ThrVal Lys Ile 35 40 45 Lys Asn Lys Asp Lys Ser Gly Asn Trp Thr Asp Leu GlyAsp Leu Val 50 55 60 Val Arg Lys Glu Glu Asn Gly Ile Asp Thr Gly Leu AsnAla Gly Gly 65 70 75 80 His Ser Ala Thr Phe Phe Ser Leu Glu Glu Glu ValVal Asn Asn Phe 85 90 95 Val Lys Val Met Thr Glu Gly Gly Ser Phe Lys ThrSer Leu Tyr Tyr 100 105 110 Gly Tyr Lys Glu Glu Gln Ser Val Ile Asn GlyIle Gln Asn Lys Glu 115 120 125 Ile Ile Thr Lys Ile Glu Lys Ile Asp GlyThr Glu Tyr Ile Thr Phe 130 135 140 Ser Gly Asp Lys Ile Lys Asn Ser GlyAsp Lys Val Ala Glu Tyr Ala 145 150 155 160 Ile Ser Leu Glu Glu Leu LysLys Asn Leu Lys 165 170 230 amino acids amino acid linear protein 3 MetAsn Lys Lys Met Phe Ile Ile Cys Ala Ile Phe Ala Leu Ile Val 1 5 10 15Ser Cys Lys Asn Tyr Thr Thr Ser Lys Asp Leu Glu Gly Ser Val Gln 20 25 30Asp Leu Glu Ser Ser Glu Gln Asn Ala Lys Lys Thr Glu Gln Glu Ile 35 40 45Lys Lys Gln Val Glu Gly Phe Leu Glu Ile Leu Glu Thr Lys Asp Leu 50 55 60Asn Thr Leu Asn Thr Lys Asp Ile Lys Glu Ile Glu Lys Gln Ile Gln 65 70 7580 Glu Leu Lys Asp Thr Ile Asn Lys Leu Glu Ala Lys Lys Thr Ser Leu 85 9095 Lys Thr Tyr Ser Glu Tyr Glu Glu Gln Ile Lys Lys Ile Lys Glu Lys 100105 110 Leu Lys Asp Lys Lys Glu Leu Glu Asp Lys Leu Lys Glu Leu Glu Glu115 120 125 Ser Leu Lys Lys Lys Lys Glu Glu Arg Lys Lys Ala Leu Glu AspAla 130 135 140 Lys Lys Lys Phe Glu Glu Phe Lys Gly Gln Val Gly Ser AlaThr Gly 145 150 155 160 Gln Thr Gln Gly Gln Arg Ala Gly Asn Gln Gly GlnVal Gly Gln Gln 165 170 175 Ala Trp Lys Cys Ala Asn Ser Leu Gly Leu GlyVal Ser Tyr Ser Ser 180 185 190 Ser Thr Gly Thr Asp Ser Asn Glu Leu AlaAsn Lys Val Ile Asp Asp 195 200 205 Ser Ile Lys Lys Ile Asp Glu Glu LeuLys Asn Thr Ile Glu Asn Asn 210 215 220 Gly Glu Val Lys Lys Glu 225 2301361 base pairs nucleic acid double linear DNA (genomic) NO NO CDS109..1359 4 AAGCCTAATT CCTTTATAGT AAGAAATAGT GCAATACATA CATTTAGTGTATGTAAAGTG 60 AGCTATATTT TTATTTAAAC CAATAATTAA ATAGAGGTAA TTTAATTT ATGAAT AAA 117 Met Asn Lys 1 ATA GGA ATT GCA TTT ATT ATT AGC TTT CTG TTGTTT GTT AAT TGT AGG 165 Ile Gly Ile Ala Phe Ile Ile Ser Phe Leu Leu PheVal Asn Cys Arg 5 10 15 GGC AAA TCT TTA GAA GAA GAT TTA AAA AGC ACC ACTTCT AAC AAT AAG 213 Gly Lys Ser Leu Glu Glu Asp Leu Lys Ser Thr Thr SerAsn Asn Lys 20 25 30 35 CAA AAT TTA ATA AGC AAT GAA AAA AAG TCT CTA AATTCT AAG AAC AAT 261 Gln Asn Leu Ile Ser Asn Glu Lys Lys Ser Leu Asn SerLys Asn Asn 40 45 50 AGG CTT AAA GAT TCT CGG TTA AGT AAT TTT GAA AGC AAAAAA AAT GAC 309 Arg Leu Lys Asp Ser Arg Leu Ser Asn Phe Glu Ser Lys LysAsn Asp 55 60 65 CAG ACA TTA AAA AAA TCC AAA GAC TTT AAA AAG GAT TTA CAAACT TTA 357 Gln Thr Leu Lys Lys Ser Lys Asp Phe Lys Lys Asp Leu Gln ThrLeu 70 75 80 AGA AAT TCA AAA AAT TTA ATG CCT AAA GAC TTG GAT CAG TCG AGTAAT 405 Arg Asn Ser Lys Asn Leu Met Pro Lys Asp Leu Asp Gln Ser Ser Asn85 90 95 GAT TTT GAA AAT TTA GAC AAT TCT GAG TCT TTG CAA GAA GCT TCT TCA453 Asp Phe Glu Asn Leu Asp Asn Ser Glu Ser Leu Gln Glu Ala Ser Ser 100105 110 115 AAG CAC AAT ATT GGC AAG TCA AGA TAC GGT AAA GCT TTG CTG AAAAAT 501 Lys His Asn Ile Gly Lys Ser Arg Tyr Gly Lys Ala Leu Leu Lys Asn120 125 130 GAT CAC GAT GAG ATT TGG ATT CCC CAT TTA AAC TTG GAA GAA GACAAA 549 Asp His Asp Glu Ile Trp Ile Pro His Leu Asn Leu Glu Glu Asp Lys135 140 145 AAT TTT GAG TTT TTC AAG AAA TCT TTG CAA AAC GAT GAG AAT AGATAT 597 Asn Phe Glu Phe Phe Lys Lys Ser Leu Gln Asn Asp Glu Asn Arg Tyr150 155 160 GCT CTT GGT GGG TGG CTT TTA AAC AAT GAT GAG GTG TTA GTA AAATAC 645 Ala Leu Gly Gly Trp Leu Leu Asn Asn Asp Glu Val Leu Val Lys Tyr165 170 175 AGA TAC AGC GAA AAA GAT GTT AAT CAG TTT TTA ATT GAT ATA GGAAAA 693 Arg Tyr Ser Glu Lys Asp Val Asn Gln Phe Leu Ile Asp Ile Gly Lys180 185 190 195 AAG CGG TGG GGA GAT TTG TCT TCT AAA ATG AGC ACC TTG GTGCGA TTG 741 Lys Arg Trp Gly Asp Leu Ser Ser Lys Met Ser Thr Leu Val ArgLeu 200 205 210 ATT GGA AAT TAT TCC GAC AAA AGT GAC AGA GAA GAT GAA ATTTCT CTT 789 Ile Gly Asn Tyr Ser Asp Lys Ser Asp Arg Glu Asp Glu Ile SerLeu 215 220 225 CTG GAT ATG AAT TTG TGT CAA CAA TTT TAT CTA ACC AAG ATTAAT GCT 837 Leu Asp Met Asn Leu Cys Gln Gln Phe Tyr Leu Thr Lys Ile AsnAla 230 235 240 GGT GGT TCA AGC GCA GAC ATT CTT GTT GCT CTT GAA AAA ACAATC GAT 885 Gly Gly Ser Ser Ala Asp Ile Leu Val Ala Leu Glu Lys Thr IleAsp 245 250 255 CAA CAA ATT AGC GGT GTT AGC AAA GAA CTT CTT GAA TTA AAAAAT TTT 933 Gln Gln Ile Ser Gly Val Ser Lys Glu Leu Leu Glu Leu Lys AsnPhe 260 265 270 275 TCT CTT ACT ACA AAG TCA GAG CTT GAT TGG TAT TTA AATTGG AAG CGC 981 Ser Leu Thr Thr Lys Ser Glu Leu Asp Trp Tyr Leu Asn TrpLys Arg 280 285 290 AAT TTA ACA GAC GAA GAA GAA GAG ACT TTG CAA TGT TGCAGG GTT TTG 1029 Asn Leu Thr Asp Glu Glu Glu Glu Thr Leu Gln Cys Cys ArgVal Leu 295 300 305 TTG GGC GGA GAA TTG GAT TTT GAA AAT CTT GAC GAT TTGTTT AAA AGG 1077 Leu Gly Gly Glu Leu Asp Phe Glu Asn Leu Asp Asp Leu PheLys Arg 310 315 320 CTT GGA AAG GAA TAT TCT AGG TTG ATA TTA AGA AAG TTAGAA GAA ATA 1125 Leu Gly Lys Glu Tyr Ser Arg Leu Ile Leu Arg Lys Leu GluGlu Ile 325 330 335 ACA TTA AAT TAC GAT GTT AAT AGG TTT TTA AAA GAA ATGGAG AAA TCA 1173 Thr Leu Asn Tyr Asp Val Asn Arg Phe Leu Lys Glu Met GluLys Ser 340 345 350 355 CGT AAA TCT TTC AAA CAA GCA TTA GGT TCT ATT AGGAAT AAA AGC AAA 1221 Arg Lys Ser Phe Lys Gln Ala Leu Gly Ser Ile Arg AsnLys Ser Lys 360 365 370 AGA GTA GTG ATT TTT AAG GTT AGA AAT TCT CTT TTGGAA ATT TTT AAA 1269 Arg Val Val Ile Phe Lys Val Arg Asn Ser Leu Leu GluIle Phe Lys 375 380 385 CTT TAT TAC AAC AAT ATT GGC AGG AAT AAA AAA CTTTAT GAT TAT ATA 1317 Leu Tyr Tyr Asn Asn Ile Gly Arg Asn Lys Lys Leu TyrAsp Tyr Ile 390 395 400 AAT CGC ATG TTA AAC AGC TTG ATA AAA GAG ATT AGCAGG CGT TA 1362 Asn Arg Met Leu Asn Ser Leu Ile Lys Glu Ile Ser Arg Arg405 410 415 417 amino acids amino acid linear protein 5 Met Asn Lys IleGly Ile Ala Phe Ile Ile Ser Phe Leu Leu Phe Val 1 5 10 15 Asn Cys ArgGly Lys Ser Leu Glu Glu Asp Leu Lys Ser Thr Thr Ser 20 25 30 Asn Asn LysGln Asn Leu Ile Ser Asn Glu Lys Lys Ser Leu Asn Ser 35 40 45 Lys Asn AsnArg Leu Lys Asp Ser Arg Leu Ser Asn Phe Glu Ser Lys 50 55 60 Lys Asn AspGln Thr Leu Lys Lys Ser Lys Asp Phe Lys Lys Asp Leu 65 70 75 80 Gln ThrLeu Arg Asn Ser Lys Asn Leu Met Pro Lys Asp Leu Asp Gln 85 90 95 Ser SerAsn Asp Phe Glu Asn Leu Asp Asn Ser Glu Ser Leu Gln Glu 100 105 110 AlaSer Ser Lys His Asn Ile Gly Lys Ser Arg Tyr Gly Lys Ala Leu 115 120 125Leu Lys Asn Asp His Asp Glu Ile Trp Ile Pro His Leu Asn Leu Glu 130 135140 Glu Asp Lys Asn Phe Glu Phe Phe Lys Lys Ser Leu Gln Asn Asp Glu 145150 155 160 Asn Arg Tyr Ala Leu Gly Gly Trp Leu Leu Asn Asn Asp Glu ValLeu 165 170 175 Val Lys Tyr Arg Tyr Ser Glu Lys Asp Val Asn Gln Phe LeuIle Asp 180 185 190 Ile Gly Lys Lys Arg Trp Gly Asp Leu Ser Ser Lys MetSer Thr Leu 195 200 205 Val Arg Leu Ile Gly Asn Tyr Ser Asp Lys Ser AspArg Glu Asp Glu 210 215 220 Ile Ser Leu Leu Asp Met Asn Leu Cys Gln GlnPhe Tyr Leu Thr Lys 225 230 235 240 Ile Asn Ala Gly Gly Ser Ser Ala AspIle Leu Val Ala Leu Glu Lys 245 250 255 Thr Ile Asp Gln Gln Ile Ser GlyVal Ser Lys Glu Leu Leu Glu Leu 260 265 270 Lys Asn Phe Ser Leu Thr ThrLys Ser Glu Leu Asp Trp Tyr Leu Asn 275 280 285 Trp Lys Arg Asn Leu ThrAsp Glu Glu Glu Glu Thr Leu Gln Cys Cys 290 295 300 Arg Val Leu Leu GlyGly Glu Leu Asp Phe Glu Asn Leu Asp Asp Leu 305 310 315 320 Phe Lys ArgLeu Gly Lys Glu Tyr Ser Arg Leu Ile Leu Arg Lys Leu 325 330 335 Glu GluIle Thr Leu Asn Tyr Asp Val Asn Arg Phe Leu Lys Glu Met 340 345 350 GluLys Ser Arg Lys Ser Phe Lys Gln Ala Leu Gly Ser Ile Arg Asn 355 360 365Lys Ser Lys Arg Val Val Ile Phe Lys Val Arg Asn Ser Leu Leu Glu 370 375380 Ile Phe Lys Leu Tyr Tyr Asn Asn Ile Gly Arg Asn Lys Lys Leu Tyr 385390 395 400 Asp Tyr Ile Asn Arg Met Leu Asn Ser Leu Ile Lys Glu Ile SerArg 405 410 415 Arg 805 base pairs nucleic acid double linear DNA(genomic) NO NO CDS 130..711 6 TCATATTAAT AAGACCTCCT GTTTCATTTTAACATTTTAA TTGTTTTTAA AGTGTGTACA 60 AAATAAATTA TTTATTGTAA ACTTACTTTTAATTTTAATA TGATTAATAA ATTATAAGGG 120 AGAATTTTT ATG TAT AAA AAT GGT TTTTTT AAA AAC TAT TTG TCA TTG 168 Met Tyr Lys Asn Gly Phe Phe Lys Asn TyrLeu Ser Leu 1 5 10 CTT TTA ATT TTT TTA GTA ATT GCT TGT ACT TCA AAA GACAGC TCA AAT 216 Leu Leu Ile Phe Leu Val Ile Ala Cys Thr Ser Lys Asp SerSer Asn 15 20 25 GAA TAT GTT GAG GAG CAA GAA GCG GAG AAC TCT TCT AAG CCTGAT GAT 264 Glu Tyr Val Glu Glu Gln Glu Ala Glu Asn Ser Ser Lys Pro AspAsp 30 35 40 45 TCT AAA ATA GAT GAA CAT ACT ATT GGG CAT GTT TTT CAC GCTATG GGA 312 Ser Lys Ile Asp Glu His Thr Ile Gly His Val Phe His Ala MetGly 50 55 60 GTA GTT CAT TCA AAA AAG GAT CGA AAA AGT TTA GGA GAA AAT ATAAAG 360 Val Val His Ser Lys Lys Asp Arg Lys Ser Leu Gly Glu Asn Ile Lys65 70 75 GTT TTT TAT TTT TCT GAA GAA GAT GGA CAT TTT CAA ACA ATA CCC TCA408 Val Phe Tyr Phe Ser Glu Glu Asp Gly His Phe Gln Thr Ile Pro Ser 8085 90 AAA GAG AAT GCA AAG TTA ATA GTT TAT TTT TAT GAC AAT GTT TAT GCA456 Lys Glu Asn Ala Lys Leu Ile Val Tyr Phe Tyr Asp Asn Val Tyr Ala 95100 105 GGA GAG GCT CCA ATT AGT ATC TCT GGA AAA GAA GCC TTT ATT TTT GTT504 Gly Glu Ala Pro Ile Ser Ile Ser Gly Lys Glu Ala Phe Ile Phe Val 110115 120 125 GGG ATT ACC TCT GAC TTT AAA AAG ATT ATA AAC AGC AAT TTA CATGGC 552 Gly Ile Thr Ser Asp Phe Lys Lys Ile Ile Asn Ser Asn Leu His Gly130 135 140 GCT AAA AGT GAT CTT ATT GGT ACT TTT AAA GAT CTT AAT ATT AAAAAT 600 Ala Lys Ser Asp Leu Ile Gly Thr Phe Lys Asp Leu Asn Ile Lys Asn145 150 155 TCA AAA TTG GAA ATT ACA GTT GAT GAG AAT AAT TCA GAT GCC AAGACT 648 Ser Lys Leu Glu Ile Thr Val Asp Glu Asn Asn Ser Asp Ala Lys Thr160 165 170 TTC CTT GAA TCT GTT AAT TAC ATT ATC GAC GGC GTT GAA AAA ATTTCA 696 Phe Leu Glu Ser Val Asn Tyr Ile Ile Asp Gly Val Glu Lys Ile Ser175 180 185 CCT ATG TTA ACG AAT TAATTTATAT TTTTGATTTT ATAGGCTTTAATCTAAATTA 751 Pro Met Leu Thr Asn 190 AAGCCTATTT TAAAAAATCA AGCTCTCAAGTCCTTTTATT AAAATTTCTG CTGT 805 194 amino acids amino acid linear protein7 Met Tyr Lys Asn Gly Phe Phe Lys Asn Tyr Leu Ser Leu Leu Leu Ile 1 5 1015 Phe Leu Val Ile Ala Cys Thr Ser Lys Asp Ser Ser Asn Glu Tyr Val 20 2530 Glu Glu Gln Glu Ala Glu Asn Ser Ser Lys Pro Asp Asp Ser Lys Ile 35 4045 Asp Glu His Thr Ile Gly His Val Phe His Ala Met Gly Val Val His 50 5560 Ser Lys Lys Asp Arg Lys Ser Leu Gly Glu Asn Ile Lys Val Phe Tyr 65 7075 80 Phe Ser Glu Glu Asp Gly His Phe Gln Thr Ile Pro Ser Lys Glu Asn 8590 95 Ala Lys Leu Ile Val Tyr Phe Tyr Asp Asn Val Tyr Ala Gly Glu Ala100 105 110 Pro Ile Ser Ile Ser Gly Lys Glu Ala Phe Ile Phe Val Gly IleThr 115 120 125 Ser Asp Phe Lys Lys Ile Ile Asn Ser Asn Leu His Gly AlaLys Ser 130 135 140 Asp Leu Ile Gly Thr Phe Lys Asp Leu Asn Ile Lys AsnSer Lys Leu 145 150 155 160 Glu Ile Thr Val Asp Glu Asn Asn Ser Asp AlaLys Thr Phe Leu Glu 165 170 175 Ser Val Asn Tyr Ile Ile Asp Gly Val GluLys Ile Ser Pro Met Leu 180 185 190 Thr Asn 32 base pairs nucleic acidsingle linear cDNA NO NO 8 TCGTGGGATC CAAGATTCAT ACTTCATATG AT 32 32base pairs nucleic acid single linear cDNA NO NO 9 GACTTCTCGA GCTATTTTAAATTCTTCTTA AG 32 32 base pairs nucleic acid single linear cDNA NO NO 10TCGTGGAATT CAAGAATTAT ACAACTAGCA AA 32 26 base pairs nucleic acid singlelinear cDNA NO NO 11 TCGAGTTATT CTTTTTTGAC TTCTCC 26

We claim:
 1. An isolated antibody that binds to a B. burgdorferipolypeptide selected from the group consisting of: (a) an OspEpolypeptide of SEQ ID NO: 2; (b) an S1 polypeptide of SEQ ID NO: 5; (c)an immunogenic fragment comprising at least 8 amino acids taken as ablock from any one of the polypeptides of (a)-(b).
 2. An isolatedantibody that binds to a B. burgdorferi polypeptide selected from thegroup consisting of: (a) an OspF polypeptide of SEQ ID NO: 3; and (b) afragment of the OspF polypeptide of SEQ ID NO: 3 from amino acid 19-230.3. A diagnostic kit comprising an antibody according to claim 1 or claim2.
 4. A method for detecting B. burgdorferi infection comprising thesteps of: (a) contacting a body fluid of a mammalian host with anantibody according to claim 1 or claim 2; and (b) detecting binding ofsaid antibody to B. burgdorferi or said polypeptide.
 5. An antibodyaccording to claim 1 or 2, wherein the antibody is monoclonal.
 6. Adiagnostic kit comprising an antibody according to claim
 5. 7. A methodfor detecting B. burgdorferi infection comprising the steps of (a)contacting a body fluid of a mammalian host with an antibody accordingto claim 5; and (b) detecting binding of said antibody to B. burgdorferior said polypetide.